Decontamination of Milk and Water by Pulsed UV-light and Infrared Heating

The Pennsylvania State University

The Graduate School

Department of Agricultural and Biological Engineering

DECONTAMINATION OF MILK AND WATER BY

PULSED UV-LIGHT AND INFRARED HEATING

A Thesis in

Agricultural and Biological Engineering

by

Kathiravan Krishnamurthy

© 2006 Kathiravan Krishnamurthy

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

August 2006

The thesis of Kathiravan Krishnamurthy was reviewed and approved* by the following: Ali Demirci Associate Professor of Agricultural Engineering Thesis Co-Adviser Co-Chair of Committee

Joseph M. Irudayaraj Associate Professor of Agricultural and Biological Engineering Purdue University

Thesis Co-Adviser Co-Chair of Committee Special Member

Virendra M. Puri Professor of Agricultural Engineering

Bhushan M. Jayarao Associate Professor of Veterinary and Biomedical Science

Roy E. Young Professor of Agricultural Engineering Head of the Department of Agricultural and Biological Engineering *Signatures are on file in the Graduate School

Abstract

Efficacy of pulsed UV-light and infrared heating for inactivation of pathogens

was investigated. Pulsed UV-light was very effective in inactivating S. aureus on agar

seeded cells and in phosphate buffer. Complete inactivation of S. aureus was achieved

within 5-s treatments.

Raw milk inoculated with S. aureus was treated with pulsed UV-light by varying

distance of milk sample from the quartz window, volume of milk, and treatment time.

The log10 reduction obtained varied from 0.16 to 8.55 log10 CFU/ml. Complete

inactivation of S. aureus was obtained at two conditions with corresponding reductions of

8.55 log10 CFU/ml.

Continuous treatment of milk was tested in order to determine the feasibility of

industrial application of pulsed UV-light treatment. Reductions of S. aurues in milk

varied from 0.55 to 7.26 log10 CFU/ml. Complete inactivation was achieved at two

conditions. Sensory evaluation of pulsed UV-light treated pasteurized skim milk and 1%

milk suggests that there was some perceivable change in the quality.

B. subtilis spores in water were treated with pulsed UV-light in an annular flow

chamber. Flow rates up to 14 L/min resulted in complete inactivation of B. subtilis

spores. No growth was observed during incubation under light and no-light conditions.

The efficacy of infrared heating on inactivation of S. aureus in milk was tested.

The effect of depth of milk, infrared lamp temperature, and treatment time were

investigated. Reductions of 0.10 to 8.41 log10 CFU/ml were obtained for treatments up to

4 min.

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The inactivation mechanism for pulsed UV-light and infrared heating was

investigated using transmission electron microscopy (TEM) and spectroscopy. After 5-s

treatment with pulsed UV-light, cell wall breakage, cytoplasm leakage, damage in the

cellular membrane structure, and leakage of the cell content were observed by TEM.

Infrared heat treated cells exhibited condensation of cytoplasm, cytoplasmic membrane

damage, cell wall damage, and cellular content leakage occurred. The FTIR spectrometry

was successfully used to classify the pulsed UV-light and infrared treated cells.

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Table of Contents

List of Figures…………………………………………………………………………….xi

List of Tables……………………………………………………………………………xiii

Acknowledgements………………………………………………………………………xv

Technical acknowledgements…………………………………………………………..xvii

1. Introduction................................................................................................................. 1

2. Literature Review........................................................................................................ 5

2.1. Milk and its microbiological significance........................................................... 5

2.2. Staphylococcus aureus........................................................................................ 6

2.3. Bacillus subtilis ................................................................................................... 7

2.4. Pasteurization of milk ......................................................................................... 8

2.5. Gamma irradiation .............................................................................................. 9

2.6. High hydrostatic pressure ................................................................................. 10

2.7. Pulsed electric fields ......................................................................................... 11

2.8. Centrifugation and membrane filtration............................................................ 11

2.9. Ultraviolet-light................................................................................................. 12

Interaction of light and matter................................................................................... 14

Inactivation mechanism of UV-light disinfection..................................................... 17

UV-light penetration and absorption ........................................................................ 20

Inactivation studies by continuous and pulsed UV-light .......................................... 23

Effect of UV on food components and quality ......................................................... 27

Water disinfection by UV-light ................................................................................ 30

Economics of UV-light disinfection system............................................................. 32

2.10. Infrared heating............................................................................................. 33

Infrared radiation basics............................................................................................ 34

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Interaction of infrared radiation with food materials................................................ 36

Inactivation mechanism of infrared heat treatment .................................................. 37

Infrared heat treatment.............................................................................................. 37

2.11. Determination of mode of microbial inactivation......................................... 42

Transmission electron microscopy ........................................................................... 42

Fourier transform infrared spectroscopy................................................................... 43

2.12. Summary of literature review ....................................................................... 44

3. Inactivation of Staphylococcus aureus by Pulsed UV-Light Sterilization ............... 47

3.1. Abstract ............................................................................................................. 47

3.2. Introduction....................................................................................................... 48

3.3. Materials and Methods...................................................................................... 52

Microorganism.......................................................................................................... 52

Sample preparation ................................................................................................... 52

Pulsed UV-light treatment ........................................................................................ 53

Microbiological analysis........................................................................................... 53

Statistical analysis..................................................................................................... 54

3.4. Results and Discussion ..................................................................................... 54

Suspended cell treatment .......................................................................................... 54

Agar seeded cells ...................................................................................................... 56

3.5. Conclusions....................................................................................................... 58

3.6. References......................................................................................................... 59

4. Inactivation of Staphylococcus aureus in Milk and Milk Foam by Pulsed UV-light

Treatment and Surface Response Modeling ..................................................................... 61

4.1. Abstract ............................................................................................................. 61

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4.2. Introduction....................................................................................................... 62


Preparation of inoculum............................................................................................ 67

Experimental design.................................................................................................. 67

Production of milk foam ........................................................................................... 68



Temperature measurements ...................................................................................... 70

Water bath studies..................................................................................................... 71

Energy measurements ............................................................................................... 71



Static milk treatment ................................................................................................. 72

Effect of sample volume ........................................................................................... 73

Effect of treatment time ............................................................................................ 74

Effect of distance ...................................................................................................... 74

Temperature profile during pulsed UV-light treatment ............................................ 76

Heat treatment of S. aureus in water bath................................................................. 77

Surface response model and validation for milk....................................................... 78

4.5. Inactivation of S. aureus in milk foam.............................................................. 81

Surface response model and validation for milk foam ............................................. 83

Energy measurement................................................................................................. 84

4.6. Conclusions....................................................................................................... 86

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4.7. References......................................................................................................... 87

5. Inactivation of Staphylococcus aureus in Milk Using Flow-through Pulsed UV-

Light Treatment System.................................................................................................... 90

5.1. Abstract ............................................................................................................. 90

5.2. Introduction....................................................................................................... 91


Preparation of inoculum............................................................................................ 94




Temperature measurement........................................................................................ 97




Surface response model .......................................................................................... 103

Temperature measurement...................................................................................... 104

5.5. Conclusions..................................................................................................... 106

5.6. References....................................................................................................... 106

6. Disinfection of Water by Flow-through Pulsed Ultraviolet Light Sterilization System

................................................................................................................................. 109

6.1. Abstract ........................................................................................................... 109

6.2. Introduction..................................................................................................... 110

6.3. Materials and Methods.................................................................................... 111

Microorganism........................................................................................................ 111

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Spore preparation .................................................................................................... 111

Pulsed UV-light treatment system .......................................................................... 114

Cleaning of the flow-through pulsed UV-light system........................................... 116

Pulsed UV-light treatment ...................................................................................... 116

Turbidity measurement ........................................................................................... 117

Radiant energy measurement .................................................................................. 117


6.4. Results and Discussion ................................................................................... 118

6.5. Conclusions..................................................................................................... 121

6.6. References....................................................................................................... 121

7. Infrared Heat Treatment for Inactivation of Staphylococcus aureus in Milk......... 123

7.1. Abstract ........................................................................................................... 123

7.2. Introduction..................................................................................................... 124


Preparation of inoculum.......................................................................................... 128

Infrared heating system........................................................................................... 128

Experimental design................................................................................................ 130

Infrared heat treatment............................................................................................ 131

Microbiological analysis......................................................................................... 131


Statistical analysis................................................................................................... 132


Effect of treatment time .......................................................................................... 135

Effect of lamp temperature ..................................................................................... 136

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Effect of volume of milk......................................................................................... 138

7.5. Conclusions..................................................................................................... 138

7.6. References....................................................................................................... 139

8. Microscopic and Spectroscopic Analysis of Inactivation of Staphylococcus aureus

by Pulsed UV-Light and Infrared Heating...................................................................... 141

8.1. Abstract ........................................................................................................... 141

8.2. Introduction..................................................................................................... 142


Inoculum preparation .............................................................................................. 144

Pulsed UV-light treatment ...................................................................................... 144

Infrared heat treatment............................................................................................ 145

TEM analysis .......................................................................................................... 145

FTIR spectroscopy analysis .................................................................................... 147


Transmission electron microscopy ......................................................................... 150

FTIR spectroscopy.................................................................................................. 157

8.5. Conclusions..................................................................................................... 165

8.6. References....................................................................................................... 166

9. Conclusions and Scope for Future Research .......................................................... 168

References………………………………………………………………………………176

Appendix A: Inactivation Kinetics of Pulsed UV-light Treatment of Staphylococcus

aureus……………………………………...……………………………………………185

Appendix B: Sensory Evaluation of Pulsed UV-light Treated Milk…………………...191

Appendix C: Source code: Infrared Heating Control Logic……………………………194

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List of Figures

Figure 2.1. Staphylococcus aureus. .................................................................................... 6

Figure 2.2. Bacillus Subtilis. ............................................................................................... 7

Figure 2.3. Germicidal efficiency of ultraviolet light. ...................................................... 17

Figure 2.4. Arrangements of lamps for flow-through systems. ........................................ 22

Figure 2.5. Radiation emissive power spectrum............................................................... 35

Figure 3.1. Log10 reductions of Staphylococcus aureus after pulsed UV-light sterilization

in suspended cell. ...................................................................................................... 55

Figure 3.2. Temperature profile of phosphate buffer and Baird-Parker agar base during

pulsed UV-light treatment......................................................................................... 56

Figure 3.3. Log10 reductions of Staphylococcus aureus after pulsed UV-light sterilization

in seeded agar plates. ................................................................................................ 57

Figure 4.1. Surface response design ................................................................................ 68

Figure 4.2. Schematic of Steripulse® XL-RS-3000 pulsed UV-light system. .................. 69

Figure 4.3. Inactivation of S. aureus in milk. ................................................................... 75

Figure 4.4. Temperature profile during pulsed UV-light treatment.................................. 77

Figure 4.5. Validation of the response surface model ...................................................... 80

Figure 4.6. Inactivation of S. aureus in milk foam. .......................................................... 82

Figure 4.7. Experimental vs. predicted log10 reduction of S. aureus in milk foam. ......... 85

Figure 5.1. SteriPulse®-XL 3000 Pulsed UV-light ........................................................... 95

Figure 5.2. Schematic diagram of continuous milk treatment system.............................. 95

Figure 5.3. V-groove reflector…………………………………………………………...96

Figure 5.4. Surface plot of log10 reduction vs. passes and distance................................ 100

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Figure 5.5. Surface plot of log10 reduction vs. flow rate and distance............................ 102

Figure 5.6. Surface plot of log10 reduction vs. flow rate and distance............................ 102

Figure 5.7. Validation of response surface design.......................................................... 104

Figure 5.8. Typical temperature profile during pulsed UV-light treatment of milk. ...... 105

Figure 6.1. Pulsed UV-light sterilization system. ........................................................... 114

Figure 6.2. Schematic diagram of cross section of the Steripulse®-RS 4000 chamber...115

Figure 7.1. Schematics of the infrared heating system…………………………………129

Figure 7.2. Lab scale infrared heating system………………………………………….129

Figure 7.3. Main effects plot (fitted means) for log10 reduction..................................... 134

Figure 7.4. Interaction plot (fitted means) for log10 reduction........................................ 135

Figure 7.5. Temperature increase during infrared heating.............................................. 137

Figure 8.1. Comparison of damages observed by TEM. ................................................ 151

Figure 8.2. Evaluation of pulsed UV-light induced damages in S. aureus by TEM. ..... 152

Figure 8.3. Microscopic evaluation of damages to S. aureus because of infrared heat

treatment………………………………………………………………………………..156

Figure 8.4. Classification of pulsed UV-light treated Staphylococcus aurues by PLS-CVA

................................................................................................................................. 158

Figure 8.5. Classification of infrared heat treated Staphylococcus aurues by PLS-CVA.

................................................................................................................................. 159

Figure 8.6. Differentiation of cells and cell walls of Staphylococcus aurues by PLS.... 160

Figure 8.7. Assignment of absorption bands for pulsed UV-light treated S. aureus. ..... 164

Figure 8.8. Assignment of absorption bands for infrared heat treated S. aureus............ 165

Figure A1. Inactivation of S. aureus by pulsed UV-light treatment. .............................. 188

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List of Tables

Table 2.1. Characteristics of UV, visible, and infrared regions of electromagnetic

spectrum.................................................................................................................... 15

Table 2.2. Percent transmission to the center of selected cells and viruses...................... 15

Table 2.3. Strength of common bonds in biomolecules.................................................... 16

Table 2.4. Photoproducts produced in DNA because of absorption of UV-light. ............ 19

Table 2.5. Wavelength dependent effective penetration depth of milk. ........................... 20

Table 2.6. Coefficient of absorption of various liquid food products at 254 nm............. 23

Table 2.7. Ultraviolet light exposure (at 254 nm) required for 4-log10 reduction of

pathogens in drinking water...................................................................................... 32

Table 4.1. Surface response method analysis of pulsed UV-light treatment of milk. ...... 73

Table 4.2. Inactivation of S. aureus by constant increase in temperature. ....................... 78

Table 4.3. Regression coefficients of response surface model for inactivation of S. aurues

in milk. ...................................................................................................................... 79

Table 4.4. Inactivation of S. aureus in milk foam. ........................................................... 81

Table 4.5. Regression coefficients of response surface model for inactivation of S. aureus

in milk foam.............................................................................................................. 84

Table 4.6. Broadband energy measurement during pulsed UV-light treatment. .............. 86

Table 5.1. Box-Behnken response surface method design parameters............................. 97

Table 5.2. Log10 reductions of S. aureus in milk by pulsed UV-light treatment. ............. 99

Table 5.3. Regression coefficients of response surface model. ...................................... 103

Table 5.4. Average bulk temperature of milk after pulsed UV-light treatment.............. 105

Table 6.1. Evaluation and comparison of spore harvesting methods. ............................ 118

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Table 6.2. Inactivation of Bacillus subtilis spores by pulsed UV-light treatment. ......... 119

Table 6.3. UV-light absorption at 254 nm. ..................................................................... 119

Table 6.4. UV-light energy measurements during pulsed UV-light treatment............... 120

Table 7.1. Log10 reduction values of S. aureus in milk by infrared heat treatment. ....... 133

Table 7.2. Analysis of variance for log10 reduction of S. aureus.................................... 134

Table 7.3. Infrared heat treatment of S. aureus for longer time...................................... 136

Table 8.1. Absorption bands of microbial cells in the infrared region ........................... 162

Table A1. Estimation of inactivation model parameters. ............................................... 188

Table A2. Sensory evaluation of UV treated milk.......................................................... 192

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Acknowledgments

I would like to express my deep gratitude and sincere appreciation to my advisors,

Dr. Ali Demirci and Dr. Joseph M. Irudayaraj for their guidance and support throughout

the course of this study. They have been a source of encouragement and provided both

academic & professional guidance. I am obliged to them for their moral and financial

support.

My sincere appreciation and profound thanks to Dr. Virendra M. Puri, Dr. Roy E.

Young, and Dr. Bhushan M. Jayarao for their guidance. Their insights and suggestions

towards my research helped me in writing this thesis.

I would like to thank my friends from our lab, Deniz Cekmecelioglu, Jamie

Bober, Katherine Bialka, Nil Ozer, Ratna Shivappa Sharma, Stephen Walker, Thunyarat

Pongtharangkul, Todd Miserendino, Yen Huang, and Zahra Lofti for their help and

friendship. Thanks to my fellow graduate students for making my experience at Penn

State memorable. Special thanks to my officemates Changying Li, Deepak Jiswal, Haiyu

Ren, Mark Bechara, Michael Nassry, Qin Chen, Xie Xinsheng for tolerating me. I would

like to thank Anand Mohan, Anand Swaminathan, Anjani Jha, Anuranjan Pandhey,

Cheng Si, Christina Torres, Deepa Naveen, Deepti Tanjore, Ebru Ersay, Jaskaran Gill,

Guoshun Zhang, Lula Ghebremichael, Mani Ammasi, Mark Corey, Mathala J. Gupta,

Matthew Lawrence, Mini Sundar, M.S. Srinivasan, Nathan Anderson, Naveen

Chikthimmah, Neetu Motwani, Nihariha Misra, Nishkaran Gunasekaran, Priya Prabhu,

Raghunath Shivappa, Rajesh Potineni, Renee Korach, Saed Sayyar Roudsari, Shankar

Radhakrishnan, Sundar Balasubramanian, Supratim Ghosh, Tanuj Motwani, and

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Vaithianathan Sundaram and others for their friendship. I am thankful to all the members

of ‘Penn State Vedic Society’ who are always there to extend their helping hands.

Thanks to my undergraduate teachers Dr. Alagusundaram, Dr. Kumar, and Dr.

Santhana Bosu for encouraging me to pursue higher studies. I am sincerely indebted to

my parents Krishnamurthy Duraiswamy and Kasturi Krishnamurthy, my brothers

Diwakaran Krishnamurthy and Ravi Krishnamurthy, Sister Jayashree Hari, Vasupratha

Diwakaran, sister-in-law Pavithra Diwakaran, and brother-in-law Hari for their concerns

and love.

xvi

Technical Acknowledgments

Funding for this project was provided in part by the Pennsylvania Agricultural

Experiment Station, USDA Special Research Grant (2002-34163-11837). Pulsed UV-

light sterilization system was provided by NASA Food Technology Commercial Space

Center equipment grant.

I am thankful to Xenon Corporation (Wilmington, MA) for the technical

assistance provided throughout the study. I particularly recognize the support provided

by Joe Peirce, Roger Williams, and Jim Lockhart. I express my sincere appreciation to

the staff at the electron microscopy facility, especially Dr. Gang Ning, Ruth Haldeman,

and Missy Hazen for their technical support. I am thankful to David Jones of Civil

Engineering department for providing support for the Total Organic Carbon analysis.

Thanks to Thomas Palchak from University creamery and Travis Edwards of Dairy

research & education center for providing the raw milk for this study. I am thankful to

Dr. Eric T. Harvill and Dr. Andrew Henderson for granting permission to use the cell

disruptor and micro-centrifuge, respectively.

I would like to express my gratitude to Dr. Soo-Jin Jun and Dr. Jagdish C. Tewari

for their technical guidance with infrared heating system and spectroscopy, respectively.

Thanks to Ruth Hollender and Julie Peterson of the sensory laboratory of Food Science

Department for their technical support in conducting sensory evaluation studies.

xvii

Dedicated to

HH Romapada Swami Maharaj

xviii

1. Introduction

Food preservation techniques have been a part of the daily life of the human race

for thousands of years because of food deterioration by pathogenic and spoilage

microorganisms. Food preservation techniques are critical for modern mass food

production and distribution. As such, various preservation technologies have been

developed and adopted successfully. Although several technologies such as, heat

treatment, cold temperature treatment, irradiation, microwave radiation, pulsed electric

field, magnetic field, high pressure, and ohmic heating treatments are available for

inactivation of these microorganisms (Juneja and Sofos, 2002), there is always a need to

investigate novel technologies as an alternative to existing preservation methods to

improve efficiency, minimize cost, and yield minimal quality changes.

In 2000, the United States produced 167.7 billion pounds of milk worth more than

$20 billion (IDFA, 2001). Milk is used in the manufacture of dairy products as well in

the manufacture of a wide variety of food products (IDFA, 2001). Improper handling and

management practices could result in contamination of the udder (Bramley and

McKinnon, 1990) and contamination of milk by pathogenic microorganisms. Hence,

effective inactivation is central to assuring milk safety. Conventional milk pasteurization

(heat treatment of milk) methods not only require a vast amount of energy, but also lack

precise control and thus incur a high capital and operational cost. In addition, during the

pasteurization process, the warm areas in the heat exchangers are conducive for the

growth of Staphylococcus aureus, a pathogenic microorganism.

In general, milk is held until it reaches the specified temperature during

pasteurization for a prolonged residence time, providing conducive environment for

1

pathogen growth.. Among these pathogens, S. aureus is the most common cause of

suppurating infections such as boils and pimples (Martin et al., 2001) and a common

cause of mastitis. According to Centers for Disease Control and Prevention (CDC), there

have been 17,248 cases of S. aureus reported during 1973-87, which accounted for 14%

of all the cases because of bacterial pathogens (Bean and Griffin, 1990; Bean et al., 1990;

Olsen et al., 2000). Therefore, it is necessary to inactivate S. aureus in food to ensure

safety of the public. Pulsed UV-light treatment and infrared heating may serve as

potential methods for inactivation of this pathogen effectively.

UV-light has been used as a bactericidal agent from as early as 1928 (Xenon,

2003). UV-light is a portion of the electromagnetic spectrum ranging from 100 to 400

nm wavelengths and has the potential to induce damage to the DNA by forming thymine

dimers (Bank et al., 1990; Miller et al., 1999). These dimers prevent the microorganism

from DNA transcription and replication, which leads to cell death (Miller et al., 1999).

UV-light can be applied in two modes, namely continuous UV-light mode and pulsed

UV-light mode. The pulsed UV-light provides higher instantaneous energy intensities

than continuous UV-light and hence is more effective in inactivating pathogens. Pulsed

UV-light is produced by accumulating the energy in a capacitor and releasing it as a short

duration pulse (in the order of nano seconds) to magnify the power greatly. UV-light

treatment provides a cost-effective alternative to the existing heat pasteurization and

preservation methods. Furthermore, taste degradation of milk subjected to UV-light

treatment is minimal, because UV-light treatment can be accomplished at an ambient

temperature (Hollingsworth, 2001).

2

Also, water is used in large quantities in the food industry for wide applications

ranging from cleaning equipments to processing/formulating food. Therefore, it is

necessary to disinfect water. Pulsed UV-light can be effectively used to inactivate

microorganisms present in water since water has a very high transmissivity for UV-light.

Continuous UV-light has been traditionally used for industrial disinfection of clean and

waste water. Bacillus subtilis is a surrogate microorganism which can be used to study

the effect of a disinfection process. As a spore former, they are resistant to many

disinfectants including UV-light. Therefore, one can assure that less resistant pathogenic

vegetative cells are inactivated completely by proving the inactivation of B. subtilis

spores.

Previous studies by several researchers indicate that pulsed UV-light is up to four

times more effective than continuous UV-light for inactivation of pathogenic

microorganisms when the total energy provided is the same. However, there is no study

done to evaluate the reasons for the enhanced effectiveness. Therefore, the effect of

pulsed UV-light on S. aureus was investigated using spectroscopy and microscopy in

order to explain the underlying inactivation mechanism. It was proposed that increased

instantaneous energy and a shocking effect because of pulsing are responsible for

increased inactivation.

Infrared radiation is part of the electromagnetic spectrum in the wavelength range

between 0.5 and 1,000 μm (Rosenthal, 1996). Though infrared heating is mainly used for

processing food materials, this approach can also be used for pathogen inactivation.

However, there are no extensive studies performed on its application for pathogen

inactivation in food systems. Several researchers have suggested numerous targets for

3

inactivation such as DNA, RNA, ribosome, cell envelope, and proteins in the cell (Sawai

et al., 1995). Far-infrared radiation can be used for heating of food systems and

inactivation of pathogens because of stronger absorption of microorganism and food

components in the far-infrared wavelength range (3 to 1,000 μm).

Therefore, the overall objective of this study was to investigate the ability of

pulsed UV-light and infrared heating as alternative technologies to inactive pathogenic

microorganisms in milk or water.

4

2. Literature Review

2.1. Milk and its microbiological significance

Milk is a biological fluid comprised of several components, such as lipids, fatty

acids, proteins, carbohydrate, salts, and other trace elements (Banks and Dalgleish, 1990).

Due to the presence of nutritious components, milk serves as a favorable medium for

microbial growth. Campylobacter jejuni, Pseudomonas spp., Xanthomonas maltophilia,

Acinetobacter spp., Moraxella spp., Neiseria spp., Kingsella spp., Ateromonas

putrefaciens, Flavobacterium spp., Alcaligenes spp., Brucella spp., Escherichia spp.,

Citrobacter spp., Salmonella spp., Enterobacter spp., Erwinia spp., Serratia spp.,

Micrococcus spp., and Staphylococcus spp. are common microorganisms associated with

milk (Gilmour and Rowe, 1990). The microbial population in milk also increases

because of mastitis, where the organisms enter the udder through the duct at the teat tip

and colonize within the udder. Some of the microorganisms such as S. aureus are

pathogenic and others are spoilage microorganisms. Therefore, it is necessary to treat

milk in order to inactivate these spoilage and pathogenic microorganisms. Traditionally,

heat pasteurization is used to achieve this goal. However, the cost associated with

pasteurization is high because of high energy demands and high capital/operational costs.

Also, S. aureus can survive because of the prolonged holding time required during the

pasteurization process. Therefore, it is necessary to find alternative processes that are

cost-effective and efficient for inactivation of S. aureus present in milk. Pulsed UV-light

and infrared heating may be used to achieve this goal.

5

2.2. Staphylococcus aureus

Staphylococcus aureus is a Gram-positive, spherical shaped bacterium, which

appears in pairs, short chains, or grape like structures (Figure 2.1). S. aureus produces

several toxins, including toxic shock syndrome toxin-1, staphylococcal enterotoxins

(SEA, SEB, SEC, SED, SEE, SEG, SHE, SEI, SEJ), exfoliative toxins (ETA, ETB), α-,

β-, δ-, and γ-hemolysins, and Panton-Valentine leukocidin (PVL) (Yarwood and

Schlievert, 2001).

Figure 2.1. Staphylococcus aureus (obtained with permission from Dennis Kunkel Microscopy, Inc., Kailua, HI).

S. aureus can cause both infection (direct ingestion of vegetative cells resulting in

multiplication in human body and production of toxins) and intoxication (ingestion of

toxins which are already produced by bacterial growth). A toxin dose level of 1 μg can

cause disease in humans. S. aureus exists in air, water, sewage, dust, human, and animals

(CFSAN-FDA, 1992). Cows affected by mastitis are a big source of S. aureus

contamination in milk. Nausea, vomiting, retching, abdominal cramping, prostration,

6

headache, muscle cramping, and transient changes in blood pressure and pulse rate are

the symptoms of S. aureus infection/intoxication. In severe cases, S. aureus can cause

death in infants, elderly, and people with weak immune system.

2.3. Bacillus subtilis

Bacillus subtilis, a Gram positive, rod shaped, spore forming soil microorganism

(Figure 2.2), though generally considered as non-pathogenic is rarely associated with

food poisoning. Food poisoning because of consumption of B. subtilis contaminated

cooked turkey, muffin, custard powder, pickled onion, mayonnaise, corn flour gel,

canned bean salad, synthetic fruit drink were reported in the past (Kramer and Gilbert,

1989).

Figure 2.2. Bacillus Subtilis (Source: Microscopy consulting services Inc., www.microscopyconsulting.com).

Severe vomiting, abdominal cramps, diarrhea are common symptoms of B.

subtilis food poisoning. B. subtilis spores are normally very resistant to several food

7

http://www.microscopyconsulting.com/

processing steps and able to survive pasteurization temperatures. Therefore, it is

necessary to inactivate the spores in order to ensure safety. B. subtilis also serves as a

surrogate organism to represent other dangerous microorganisms of the same genus such

as B. cereus and B. anthracis. As these microorganisms are genetically similar, a process

which inactivates Bacillus subtilis may also effectively inactivate pathogenic B. anthracis

and B. cereus.

The following sections, namely, irradiation, high hydrostatic pressure, pulsed

electric field, centrifugation, membrane filtration, UV-light treatment, and infrared

heating will discuss conventional pasteurization techniques and technologies for the

inactivation of S. aureus, B. subtilis and/or other pathogens.

2.4. Pasteurization of milk

Conventionally, heat treatment is used for pasteurization of milk. For

pasteurization, milk is held at 62.8oC for 30 min (low temperature-long time: LTLT),

71.7oC for 15 s (high temperature-short time: HTST), or 137.8oC for 2 s (Ultra high

temperature: UHT) (Varnam and Sutherland, 1994; Zall, 1990). Milk is tested for the

presence of alkaline phosphatase in the pasteurized milk in order to ensure that milk has

been adequately pasteurized (Cornell, 1998). Alkaline phosphatase has higher heat

stability than the pathogens present in the milk and hence used as an indicator of ensuring

adequate pasteurization. Normally milk is preheated in the regenerator section of the

pasteurization unit in order to increase the thermal efficiency and save energy. The

preheated milk flows through a heat exchanger, which consists of series of plates in

which the milk and the heating medium are moving counter-currently. The milk passes

8

through the holding tube, where the milk is kept at the desired temperature for the

minimum specified time (Varnam and Sutherland, 1994). Though pasteurization is

effective in inactivating microorganisms, the method has many disadvantages.

Nutritional quality of milk decreases during heat pasteurization processing and storage,

losses in vitamins, proteins, lactose, and minerals especially occur (Varnam and

Sutherland, 1994). Heat pasteurization also affects the structure and quality of milk

because of protein denaturation, enzyme inactivation, lipid oxidation, and non-enzymatic

browning. Campylobacter spp., L. monocytogenes, Yersinia enterocolitica, S. aureus,

and Bacillus cereus may be present in pasteurized milk because of underprocessing and

post-processing contamination (Varnam and Sutherland, 1994). Warm spots are created

in the equipment because of prolonged residence time. The warm spots may promote

microbial growth by providing a conducive environment. Therefore, there is a need for

other innovative techniques to ensure the safety of milk. Among several alternative

techniques available, irradiation, high hydrostatic pressure, pulsed electric field,

centrifugation, membrane processing, pulsed UV-light treatment, and infrared heating are

discussed in the following sections.

2.5. Gamma irradiation

Gamma radiation has a very high energy level and can disrupt even the most

stable atom, which in turn produce ions and free radicals. These free radicals damage the

DNA and RNA of bacterial cells leading to cell death (Graham, 1992). The effects of

irradiation on biological materials can be direct, where the energy is directly utilized in

breakage of chemical bonds in DNA or RNA; or indirect, wherein the free radicals

9

produced ionize water and other food components and produce further radicals, which in

turn result in cell damage. Thus the free radicals damage of DNA or RNA molecules is

hypothesized as a major destruction mechanism by irradiation (Desrosier and Desrosier,

1977). Irradiation dose is expressed in kiloGray (kGy) (The amount of energy equivalent

to 1 kJ/kg absorbed by a material from ionizing radiation is equivalent to 1 kGy). The

D10 values of S. aureus and B. cerus spores are 0.26-0.6 and 1.6 kGy, respectively

(Farkas, 2001). Generally milk and many dairy products are unsuitable for irradiation

because of increased quality changes (Crawford and Ruff, 1996).

2.6. High hydrostatic pressure

High Hydrostatic Pressure (HHP) treatment is the application of high hydrostatic

pressure in the range of several hundred Mega Pascals (MPa) on the food normally

submerged in liquid. High hydrostatic pressure has the following effects on

microorganisms: 1) cell envelope-related effects, 2) pressure induced cellular changes, 3)

biochemical aspects, and 4) effect on genetic mechanisms (Hoover, 2001). These

changes are because of increased pressure and altered cellular morphology. The cell

division slows down because of HHP because of the altered cellular morphology

(Hoover, 2001). HHP is very effective in pathogen reduction as demonstrated by several

researchers. For example, Patterson et al. (1995) obtained 5 log10 CFU/ml reduction of S.

aureus at 700 MPa pressure when treated for 15 min.

10

2.7. Pulsed electric fields

Pulsed Electric Fields (PEF) involve high voltage electric pulses, which are very

effective in inactivating pathogens (Molina et al., 2002). PEF induce electroporation and

disruption of semi-permeable membranes leading to breakdown of organelles by swelling

or shrinking (Castro, 1993). The main inactivation mechanism is the increase in cell

permeability because of compression and poration (Vega-Mercado et al., 1996). Several

researchers demonstrated that PEF is effective in inactivation of several pathogens. For

instance, a 9 log10 reduction of E. coli suspended in simulated milk ultrafiltrate was

obtained by Zhang et al. (1994) when a converged electric field intensity of 70 kV/cm

was applied for 160 μs. S. aurues population in skim milk was reduced by 3.7 log10

CFU/ml when exposed to 3.5 kV/mm electric field strength for 450 µs when a stepwise

mode fluid transmission system was used (Evrendilek et al., 2004).

2.8. Centrifugation and membrane filtration

Bacterial removal by centrifugation can be utilized in milk processing. Removal

of bacteria and bacterial spores by high speed centrifugation is called bactofugation. It is

commonly used in Europe. Centrifugation is a highly energy demanding process

typically resulting in 90-95% reduction of bacterial spores (Guerra et al., 1977). Su and

Ingham (2000) reported that bacterial spores can be effectively removed from milk by

this process. Spores of Clostridium tyrobutyricum,Clostridium butyricum, Clostridium

sporogenes, and Clostridium beijerinckii in skim milk were removed by subjecting the

milk to various centrifugal forces ranging from 3,000 x g to 12,000 x g. Spore reductions

of more than 97% were achieved when skim milk was centrifuged at 12,000 x g for 30

11

seconds for the abovementioned microorganisms. As the centrifugal force increased,

spore removal also increased.

Several membrane filtration techniques such as microfiltration and ultrafiltration

can be utilized for cold pasteurization of milk. This preserves the milk quality because of

the absence of heat induced quality changes. Microfiltration is much more effective than

centrifugation for removal of bacteria and bacterial spores (Brans et al., 2004).

Clostridium tyrobutyricum and Bacillus cereus spores were reduced by more than 5log10

CFU/ml when the permeate flux of 400 kg/h/m2 was used for 150 min in a reverse

asymmetric membrane with average pore size of 0.87 µm (Guerra et al., 1997). Grant et

al. (2005) achieved up to 95% reduction of Mycobacterium avium sbsp. paratuberculosis

in whole milk by centrifugation of pre-heated milk at 60oC at 7000 x g for 10 s or

microfiltration through a filter of 1.2 µm pore size.

2.9. Ultraviolet-light

Ultraviolet (UV) light has been used as a bactericidal agent from as early as 1928

(Xenon, 2003). UV-light is divided into four regions namely UV-A (315-400 nm), UV-B

(280-315 nm), UV-C (200-280 nm), and vacuum UV (100 to 200 nm) according to their

wavelength (Perchonok, 2003). UV-light can be applied in two modes namely

continuous mode and pulsed mode. In continuous mode, constant energy UV-light is

released continuously in a monochromatic or polychromatic wavelength. In the pulsed

mode, the electrical energy is stored in a capacitor over a short period of time (few

milliseconds) and released as very short period pulses (several nanoseconds). The

electrical energy is transferred through a lamp filled with inert gas (xenon or krypton),

12

which causes ionization of gas and produces a broad spectrum of light in the wavelength

region of ultraviolet to near infrared. The intensity of pulsed light is 20,000 times more

than that of sunlight (Dunn et al., 1995). Typically the pulse rate is 1 to 20 pulses per

second and the pulse width is 300 ns to 1 ms. Therefore, light pulses with high energy in

several Megawatts are produced though the total energy is comparable to continuous UV-

light system. Pulsed UV-light treatment is a more effective and rapid way of inactivating

the microorganisms than continuous UV-light sources because the energy is multiplied

many fold (Dunn et al., 1995).

Pulsed light is a broad spectrum radiation from UV-light to infrared radiation.

Pulsed light is also referred to pulsed UV-light, high intensity light, UV-light, broad-

spectrum white light, pulsed white light, and near infrared light (Green et al., 2003).

Typically, the wavelength of pulsed light ranges from 100 to 1100 nm. As the majority

of the energy was received from UV-light portion in this study, the term pulsed UV-light

was used throughout this thesis (54% Ultraviolet, 26% Visible, and 20% Infrared; Xenon,

2003). The UV portion of the pulsed UV-light has higher energy level followed by

visible light and infrared region (Table 2.1).

Previous research shows that pulsed UV-light is at least two times effective as the

continuous UV-light (McDonald, 2000). Pulsed UV-light may have some shocking

effect on the cell wall of bacteria in addition to the effect of high intensity pulses. But

more research has to be done before arriving at a conclusion. Localized heating of

bacteria is induced by pulsed UV-light as the heating and cooling rate of bacteria and the

surrounding matrix is different (Fine and Gervais, 2004). Pulsed UV-light is gaining

attention in recent years, because it can provide sufficient antimicrobial inactivation and

13

commercial sterilization with no toxic by-products (FDA, 2000). It can be effectively

used to inactivate pathogens on the surface of food or packaging materials. Furthermore,

it can also be used for in-package sterilization if a packaging material can allow UV-light

to penetrate (Butz and Tauscher, 2002).

Interaction of light and matter

Light consists of discrete fundamental packets of energy known as photons which

have zero mass, no electric charge, and an indefinitely long lifetime. These photons

contain vast amount of energy, which is determined by the wavelength of light (Equation

2.1). For instance, UV-light photon at 254 nm has energy of 470 kJ/mol (Table 2.1).

λυ hc== h E (Equation 2.1)

where, E is the energy of photon, h is the Planck’s constant (6.626 x 10-34 J.s), υ is the

frequency of light, c is the speed of light in vacuum, and λ is the wavelength of light.

Typical quantum energy of photons is given for the region of pulsed UV-light in

Table 2.1. Photons in ultraviolet region have more energy (Table 2.1) than visible or

infrared region and hence it accounts for predominant inactivation of pathogens.

When a pulsed light of initial intensity (Io) falls on food surface, portion of the

light is transmitted through the food, while the rest is reflected back and/or scattered. As

the pulsed UV-light penetrates through the food material, its intensity decays along a

distance of x beneath the food surface (Palmieri et al., 1999) given by

XOeTII −= (Equation 2.2)

where, T is the transparency coefficient of the food material, I is the intensity of light at a

distance x from the surface, Io is the initial intensity of light, and x is the distance below

14

the food surface. The residual amount of light is dissipated as heat and transferred to the

inner layers through conduction (Palmiri et al., 1999). Therefore, the intensity of UV-

light exponentially decays within the food material. UV-light is more effective for

surface sterilization and sterilization of highly transparent liquids such as water.

Table 2.1. Characteristics of UV, visible, and infrared regions of electromagnetic

spectrum.

Region Wavelength (nm) Frequency (Hz) Photon energy

(eV) Molar photon

energy (kJ/mol) Vacuum UV 100 – 200 3.00x1016 to 3.00x1015 124 to 12.4 11975 to 1197

UV-C 200 – 280 3.00x1015 to 1.07x1015 12.40 to 4.43 1197 to 427 UV-B 280 – 315 1.07x1015 to 9.52x1014 4.43 to 3.94 427 to 380 UV-A 315 – 400 9.52x1014 to 7.49x1014 3.94 to 3.10 380 to 299

Visible light 400 – 700 7.49x1014 to 4.28x1014 3.10 to 1.77 299 to 171 Near Infrared 700 – 1400 4.28x1014 to 2.14x1014 1.77 to 0.89 171 to 85.5 Mid infrared 1400 – 3000 2.14x1014 to 9.99x1013 0.89 to 0.41 85.5 to 39.9 Far infrared 3000 -10000 9.99x1013 to 3.00x1013 0.41 to 0.12 39.9 to 12.0

Less transparent foods have to be treated in a thin layer to overcome the

penetration limitation. Also for liquid foods, good mixing aids in uniform exposure.

UV-light is absorbed and penetrates into the microorganism depending upon the chemical

composition, size of the microorganism, wavelength of interest, and medium of

introduction etc. (Table 2.2).

Table 2.2. Percent transmission to the center of selected cells and viruses*.

Percent transmission at selected wavelengths (%)Biological

sample Diameter

(µm) 200 nm 250 nm 300 nm 350 nm Virus (Herpes

simplex) 0.15 66 80 100 100

Bacteria 1 33 78 98 100 Yeast 5 1.6 69 97 100

*Coolhill, 1995.

15

Table 2.3 lists the chemical bond energy of some common chemical bonds. It is

clear that UV-light has sufficient energy to break most of the chemical bonds. Hence,

ultraviolet light can cause cleavage in organic compounds. Ultraviolet light has the

energy in the magnitude of covalent bond energy, thus it mainly breaks the covalent

bonds of the target material.

Table 2.3. Strength of common bonds in biomolecules*.

Chemical Bond

Type Wavelength Bond dissociation energy (kJ/mole)

N≡N 129 930 C≡C 147 816 C=O 168 712 C=N 195 615 C=C 196 611 P=O 238 502 O-H 259 461 H-H 275 435 P-O 286 419 C-H 289 414 N-H 308 389 C-O 340 352 C-C 344 348 S-H 353 339 C-N 408 293 C-S 460 260 N-O 539 222 S-S 559 214

* Nelson and Cox, 2000.

As the energy of photons in UV-light range is high they can even cause ionization

of molecules, whereas visible light and infrared region causes vibration and rotation of

molecules, respectively. When molecules absorb the energy, it is elevated to an excited

state. The excited molecule can either 1) relax back to the ground state by releasing the

16

energy as heat, 2) relax back to the ground state by releasing energy as photons, or 3) can

induce some chemical changes.

Inactivation mechanism of UV-light disinfection

UV-light exhibits germicidal properties in the wavelength range of 100 to 280 nm

(UV-C region). The inactivation efficiency follows a bell shaped curve where maximum

inactivation occurs approximately at 254 to 264 nm range (Figure 2.3). Therefore, it is

crucial to design a disinfection system, which emits higher intensities of wavelengths in

the absorption region of interest. However, typical mercury UV lamps can deliver at 254

nm maximum. Therefore, it is usually mentioned that UV inactivation is at 254 nm as

shown in Figure 2.3.

Figure 2.3. Germicidal efficiency of ultraviolet light (Masschelein, 2002).

As only the absorbed UV-light energy induces photophysical, photochemical,

and/or photothermal effect necessary for inactivation of pathogenic microorganisms, it is

17

crucial to have a proper lamp design. Base pairs of DNA absorb UV-light effectively

because of their aromatic ring structure. In general, pyrimidines (Thymine (DNA),

cytosine (DNA and RNA) and Uracil (RNA)) are strong absorbers of photons in the

Ultraviolet range and thus produce photoproducts which results in bacterial inactivation

(Masschelein, 2002).

UV-light damages the DNA of bacteria by primarily forming thymine dimers and

thus bacterial DNA cannot be unzipped for replication. Hence bacteria cannot reproduce

(Bank et al., 1990; Jay, 2000; Demirci, 2002). Though cyclobutyl pyrimidine dimer

formation is the main inactivation mechanism, there are other photoproducts formed

during UV-light exposure including pyrimidine pyrimidinone [6-4]-photoproduct, Dewar

pyrimidinone, Adenine-thymine heterodimer, cytosine photohydrate, thymine

photohydrates, single strand break, and DNA-protein crosslink (Table 2.4). Setlow and

Setlow (1962) reported that DNA chain breakage, cross-linking of strands, hydration of

pyrimidines, and formation of dimers between adjacent residues in the polynucleotide

chain are the major photochemical changes that occur in DNA upon UV-light exposure.

Formation of photoproducts depends on wavelength, DNA sequence, and protein-DNA

interactions (Mitchell, 1995). Aromatic amino acids such as phenylalanine and

tryptophane also absorb ultraviolet light (Henis, 1987) and thus inactivates these amino

acids present in microorganisms.

18

Table 2.4. Photoproducts produced in DNA because of absorption of UV-light*.

Percentage of total photoproducts Photoproduct UV-C UV-B Cyclobutyl pyrimidine dimer 77 78 Pyrimidine pyrimidinone[6-

4]-photoproduct 20 10

Dewar pyrimidinone 0.8 10 Adenine-thymine heterodimer 0.2 -

Cytosine photohydrate ~2.0 ~2.0 Thymine photohydrates n/a n/a

Single strand break <0.1 <0.1 DNA-protein cross link <0.1 <0.1 *Mitchell, 1995.

Photoreactivation occurs in the wavelength range of 330 to 480 nm because of

activation of DNA photolyase, which splits thymine dimers (Walker, 1984). It is also

hypothesized that thermal stress induced on bacteria because of UV-light exposure leads

to bacterial rupture especially at higher flux densities (0.5 J/cm2). Overheating of

bacteria is caused because of differences in UV-light absorption by bacteria and

surrounding medium and thus bacteria becomes a local vaporization center and may

generate a small steam flow performing membrane destruction (Takeshita et al., 2003).

UV-A (315 to 400 nm) has better penetration capacity than UV-C and affects

bacterial cells by causing membrane damages and/or generates active oxygen species or

H2O2 (Kramer and Ames, 1987). However, UV-A has very little impact on microbial

cells unless exogenous photosensitizers are used in the process and absorbed by the

bacterial cell (Mitchell, 1978). Antimicrobial effect of pulsed UV-light is predominately

caused by the UV-light portion of the broad spectrum.

The inactivation mechanism for spores is different from that of vegetative cells

mainly because of the structural differences. Riesenman and Nicholson (2000) reported

19

that the thick protein coat induces resistance in spores of Bacillus subtilis. The DNA of a

bacterial spore has a different conformation than the DNA of the vegetative cell. Setlow

and Setlow (1987) reported that Bacillus species spores did not produce any detectable

amount of thymine containing dimers. The predominant photoproduct was 5-thyminyl-

5,6 dihydrothymine adduct; later it was termed as ‘spore photo-product’.

UV-light penetration and absorption

UV-light can penetrate only up to several millimeters in food material depending

upon the optical properties of the food materials. UV-light can easily penetrate through

water since it is transparent to the wavelengths produced by pulsed UV-light. Foods such

as sugar solutions have limited penetration. The effective penetration depth for milk

(2.7% fat content and 3.3% protein content) was reported by Burton (1951) (Table 2.5).

As milk is opaque, UV-light cannot penetrate well hence milk has to be presented to the

system as thin layer.

Table 2.5. Wavelength dependent effective penetration depth of milk*.

Effective penetration depth (mm) Wavelength (nm)

250 0.036 275 0.038 300 0.041 400 0.050 500 0.058 600 0.065 700 0.073 800 0.080

* Burton, 1951.

20

Oppenlander (2003) suggested some of the possible arrangements of the UV-light

lamp in a photo-reactor, which can be utilized to enhance the effectiveness of absorption

by target material (Figure 2.4). A falling film photo-reactor design might be essential for

effective penetration in milk. Other designs which reduce the thickness of milk to be

treated would be crucial for the commercial success of this technology for use in opaque

liquid foods. The designs proposed by Oppenlander (2003) can be extended to other

liquid foods and water disinfection. Addition of some absorption enhancing agents such

as edible colorants will also enhance UV-light penetration (Palmieri et al., 1999).

Another approach is to combine UV-light with other technologies such as infrared

heating to achieve the desired microbial reduction. The combined treatment of UV-light

and infrared heating resulted in considerable reduction in the microflora of milk (Giraffa

and Bossi, 1984). For instance, a 40oC infrared heat treatment combined with 45.6 s UV-

light treatment at 2000 L/h flow rate of milk resulted in reductions of standard plate

count, Lactic acid bacteria, enterococci, coliforms, psychrotrophic bacteria, and spores by

92%, >99%, 70%, 95%, 99%, and 53%, respectively.

21

Figure 2.4. Arrangements of lamps for flow-through systems (Oppenlander, 2003).

Cross sectional and top-view of arrangements. A: continuous flow annular photoreactor with coaxial lamp position, B: External lamp position with reflector (R), C: Perpendicular lamp position, D: Contact-free photoreactor types (including falling film, flat bed). L: Lamp, RV: reactor vessel, Q: Quartz tube.

The absorption coefficient of liquid food increases as the color or turbidity of

liquid increases. The penetration capacity of UV-light reduces as the absorption

coefficient increases (Guerrero-Beltran and Barbosa-Canovas, 2004). Shama (1999)

reported the coefficient of absorption for various liquid foods (Table 2.6). Therefore, for

UV-light treatment of foods with higher coefficients of absorption, optimization of the

system which will enhance the penetration depth will be beneficial. Furthermore,

treatment of these food products in the form of a thin film may result in better penetration

and thus increase the efficiency of microorganism inactivation.

22

Table 2.6. Coefficient of absorption of various liquid food products at 254 nm*.

Coefficient of absorption (cm-1) Liquid food product

Distilled water 0.007-0.01 Drinking water 0.02-0.1

Clear syrup 2-5 White wine 10 Red wine 30

Beer 10-20 Dark syrup 20-50

Milk 300 * Shama, 1999.

Inactivation studies by continuous and pulsed UV-light

McDonald et al. (2000) compared the effectiveness of a continuous UV-light

source and a pulsed UV-light source for the decontamination of surfaces and reported an

almost identical level of inactivation of B. subtillis with 4x10-3 J/cm2 of pulsed UV-light

source and 8 x10-3 J/cm2 continuous UV-light source.

Chang et al. (1985) investigated the effect of UV-light for the inactivation of

Escherichia coli, S. Typhi, Shigella sonnei, Streptococcus faecalis, and S. aureus. The

bacteria were grown in nutrient broth at 35oC for 20 to 24 h. The culture was then

filtered with a 0.45 μm filter and rinsed with sterile buffer water. Cells were resuspended

in sterile buffer water and the aggregated groups of bacteria were removed using a 1.0

μm nucleopore polycarbonate membrane. After the treatment of filtrate with UV-light,

samples were grown in nutrient agar and the bacterial colonies were enumerated. E. coli,

S. aureus, S. Sonnei, and S. Tyhi exhibited similar resistance to the UV-light and a 3log10

reduction was obtained with approximately 7 x10-3 J/cm2 energy. However, the

23

resistance exhibited by S. faecalis was higher and required a 1.4 times higher dose than

the above-mentioned microorganisms to get 3 log10 reduction of inactivation.

The effect of pulsed UV-light emission with low or high UV content on

inactivation of L. monocytogenes, E. coli, S. Enteritidis, Psudeomonas aeruginosa, B.

cerus, and S. aureus were investigated by Rowan et al. (1999). Bacterial cultures were

seeded separately on the surface of Tryptone soya-yeast extract agar and treated with a

pulsed UV-light source with low or high UV content. Reductions of 2 and 6 log10 were

obtained with 200 light pulses (approximate pulse duration = 100 ns) for low and high

UV content, respectively. Sonenshein (2003) investigated the effect of high-intensity UV

light on inactivation of B. subtilis spores. B. subtilis spores were diluted to give

concentrations of 1x109 (Sample A), 1x108 (Sample B), or 1x107 (Sample C) using sterile

deionized water. A 50 μl sample of each concentration were placed at three different

positions (Position 1: on the lamp axis and at the midpoint of the lamp; Position 2: 1 cm

above the lamp axis and at the midpoint of the lamp; Position 3: 1 cm above the lamp

axis and 172 mm to the right of the midpoint of the lamp) and treated with pulsed UV-

light. Three pulses (1 s) of UV-light resulted in more than 6.5 log10 CFU/ml (Sample B)

and 5.5 log10 CFU/ml (Sample C) reductions when the samples were placed at the lamp

axis and at the midpoint of the lamp.

Stermer et al. (1987) investigated the effect of UV radiation for inactivation of

bacteria in lamb meat. With 4 x10-3 J/cm2 energy, 99.9% of the naturally occurring floras

of lamb (mostly Pseudomonas, Micrococcus, and Staphylococcus spp.) were inactivated.

The bactericidal effectiveness of modulated UV-light was investigated by Bank et al.

(1990). Bacterial monolayers of S. epideridis, Pseudomonas aeruginosa, E. coli, S.

24

aureus, or S. marcescens on Trypticase soy agar (TSA) plates were exposed to modulated

pulsed UV-light. A 60 s treatment time at 31 cm distance from the light source

(approximately 4 x10-4 J/cm2) resulted in 6 to 7 log10 reduction of viable bacterial

population.

The effect of continuous ultraviolet energy to inhibit the pathogens on fresh

produce was investigated by Yaun et al. (2004). The surfaces of red delicious apples, leaf

lettuce, and tomatoes were inoculated with Salmonella spp. or E. coli O157:H7 and

treated with UV-C light at a wavelength of 253.7 nm with different doses ranging from

1.5 to 24x10-3 W/cm2. A 3.3 log10 CFU/apple reduction was obtained for E. coli

O157:H7 at 24x10-3 W/cm2, whereas, a 2.19 log10 CFU/tomato reduction was obtained

for E. coli O157:H7 at 24x10-3 W/cm2. Similarly, lettuce inoculated with Salmonella

spp. and E. coli O157:H7 resulted in 2.65 and 2.79 log10 CFU/lettuce reductions,

respectively.

Takeshita et al. (2003) investigated the mechanisms of damage of yeast cells

induced by pulsed light and continuous UV-light. The authors reported that the DNA

damage induced by continuous UV-light is slightly higher than that of pulsed light.

Protein elution because of pulsed UV-light was higher than that resulting from

continuous UV-light. Wekhof et al. (2000) suggested that the inactivation mechanism of

pulsed UV-light includes both the germicidal action of UV-C light and rupture of

microorganism because of thermal stress caused by UV components.

Aspergillus niger spores in corn meal were inactivated using a pulsed UV-light

(Jun et al., 2003). A 4.95 log10 reduction of A. niger on inoculated corn meal with a

treatment time of 100 s was achieved when the distance between the quartz window and

25

sample was kept at 8 cm and the input voltage was 3800 V (Jun, 2003). Sharma and

Demirci (2003) investigated the effect of pulsed UV-light on inactivation of E. coli

O157:H7 on alfalfa seeds. Reductions of 0.09 to 4.89 log10 CFU/g were obtained for

various thickness and treatment time combinations. Four thicknesses (1.02, 1.92, 3.61,

and 6.25 mm) and 7 treatment times (5, 10, 30, 45, 60, 75, and 90 s) were used in the

experiment. Complete inactivation of E. coli O157:H7 was obtained within 30 s

treatment time when the seed thickness was maintained at 1.02 mm, which corresponds

to 4.80 log10 CFU/g. An increase in treatment time resulted in a higher log10 reduction

for all the thicknesses (p<0.05). This can be concluded since more than 4.0 log10 CFU/g

reduction was obtained at the highest treatment time for all the thicknesses. Hillegas and

Demirci (2003) studied the effect of pulsed UV-light on inactivation of Clostridium

sporogenes in honey. An 87.6% reduction of Clostridium sporogenes was obtained when

a 2 mm depth of honey sample was kept at 8 cm distance from the quartz window and

treated with UV-light for 45 s (initial inoculum level was 6.24 log10 CFU/g); whereas,

180 s treatment time was required to achieve 89.4% reduction at 20 cm distance from the

quartz window.

Smith et al. (2002) used a pulsed laser excimer source to created pulsed UV-light

at 248 nm to treat bulk tank bovine milk. After exposed to 25 J/cm2 energy (114 s

exposure), there was no growth observed for cultures of Escherichia coli O157:H7,

Listeria monocytogenes, Salmonella Dublin, Yersinia enterocolitica, Staphylococcus

arueus, Aeromonus hydrophillia, and Serratia marcescens. The corresponding microbial

reduction is more than 2 log10 CFU/ml. UV-light can also degrade toxins. Yousef and

Marth (1985) reported that upon UV-light exposure, reductions of 3.6 to 100% of

26

alfatoxin M1 was achieved in milk with treatments of 2 to 60 min. Pulsed UV-light

inactivation depends on several factors such as, transmissivity of the food product,

geometry of the waveguide, lamp power, and exposed wavelength range.

Effect of UV on food components and quality

In the presence of air, depolymerization of starch occurs under UV-light

(Tomasik, 2004). Presence of sensibilizers (metal oxides, particularly ZnO) enhances

this process. Usually, photooxidation followed by photodecomposition occurs. UV-light

exposure initiates free radical oxidation and catalyzes other stages of the process. UV-

light forms lipid radicals, superoxide radicals (SOR), and H2O2 (Kolakowska, 2003).

SOR can further induce carbohydrate crosslinking, protein crosslinking, protein

fragmentation, peroxidation of unsaturated fatty acid, and loss of membrane fluidity

function. UV radiation may also denature proteins, enzymes, and amino acids (especially

amino acids with aromatic compounds) in milk, leading to textural changes. Water also

absorbs UV photons and produces OH- and H+ radicals, which in turn aids changes in

other food components. Therefore, UV-light treatment not only changes the chemistry of

food components, but also leads to product quality deterioration when it is applied at high

doses. However, most of the changes in the components are detrimental to microbial

growth. Therefore, proper optimization of the disinfection process is necessary in order

to maintain the quality of food products while ensuring its safety. Normally, microbial

inactivation can be achieved within seconds to minutes depending upon the opacity of the

food products and microorganism type.

27

UV-light may cause off flavors and color changes in food (Ohlsson and

Bengtsson, 2002). During UV-light treatment, oxygen radicals are formed which leads to

formation of ozone, especially between 185 to 195 nm, which causes off-flavors in milk.

In the wavelength region of 280 to 310 nm, cholesterol (provitamin) changes into natural

vitamin D3 and hence enriches milk with vitamin D. UV-light exposure was used for

vitamin D enrichment in milk in early days until vitamin D production became cheaper.

UV-light enrichment of other products can also be achieved by ultraviolet light exposure.

Jasinghe and Perera (2006) successfully fortified edible mushrooms with Vitamin D2.

The Shitake, oyster, button and abalone mushrooms were treated with UV-A (315-400

nm), UV-B (290-315 nm), and UV-C (190-290 nm) for a period of up to 1 h on each side

of the mushrooms. The vitamin D2 content ranged from 22.9±2.7 to 184.0±5.7 for

various mushroom and UV-light combinations. The authors noted that even treatment of

5 g of shitake mushrooms for 15 min with UV-A or UV-B can provide more than the

recommended allowance of vitamin D for adults (10 µg/day).

Light induced flavor is caused because of activation of riboflavin, which is

responsible for the conversion of methionine from methanol which leads to a burnt-

protein like, burnt-feathers like, or medicinal-like flavor. Peroxides produced during UV-

light exposure may also attack fat soluble vitamins and colored compounds and may lead

to change in nutritional quality and/or discoloration. Furthermore, long treatments with

UV-light may increase the temperature of food product which will lead to temperature

related quality changes such as cooked flavor, change in color because of non-enzymatic

browning. UV-light can also degrade vitamins by photodegradation. Especially vitamins

A, C, and B2 are affected by UV-light.

28

Prolonged treatment with UV-light can result in discoloration of food materials.

Cuvelier and Berset (2005) reported that paprika gels started to fade upon UV-light

exposure after 3 h. Fat soluble vitamins and colored compounds can be affected by the

peroxides produced during extended UV-light treatment. Undesirable changes in food

may occur when food is treated with UV-light for an extended period of time. Foods

may need to be treated for less time to achieve the desired decontamination level, and

hence there will not be any adverse change in food quality.

The in-package UV-light treatment of white bread slices with the PureBright®

system resulted in bread slices with a fresh appearance for more than two weeks,

however, the control slices were dried out and got moldy (Rice, 1994). The author also

suggested that the quality of tomatoes treated with pulsed light was acceptable up to 30

days in storage at refrigerated temperature.

Choi and Nielsen (2005) investigated the efficacy of UV-light for pasteurization

of apple cider since FDA has approved the use of UV-light for reduction of pathogens in

fruit and vegetable juices (Federal register, 2000). The consumer panel sensory

evaluation with 40 panelists suggested that there was no significant difference (p<0.05)

for the odor, color, cloudiness, sweetness, acidity, overall flavor, and overall product

ratings for untreated, pasteurized, and UV-light treated apple ciders stored for 4 days.

Furthermore, the pasteurized apple cider received less acceptable rating for color,

cloudiness, and overall product preference than control and UV-light treated samples.

This clearly indicates that the quality changes are minimal in UV-light treated apple cider

at optimum conditions when compared to thermal pasteurized cider. Consumer panelists

significantly preferred UV-light treated apple cider over thermally pasteurized apple cider

29

for color, cloudiness, sweetness, acidity, overall flavor, and overall likeness than

pasteurized cider (Choi and Nielsen, 2005).

The efficacy of pulsed UV-light for decontamination of minimally processed

vegetables was investigated by Gomez-Lopez et al. (2005). They conducted a sensory

evaluation procedure with a semi-trained panel of 4 to 6 people. The panelists evaluated

the quality of minimally processed white cabbage and iceberg lettuce. Off-odors were

present for the pulsed light treated white cabbage just above the acceptable limit. This

limited the shelf life of white cabbage to a maximum of seven days. The panelists

described the off-odor as “plastic”, which was distinctive immediately after the pulsed

light treatment, but the off-odor faded away after a couple of hours in storage. Therefore,

the off-odor can be assumed to disappear before consumption by the consumers. It is

interesting to note that pulsed UV-light treated iceberg lettuce received better scores than

the control samples for off-odor, taste, and leaf edge browning. This clearly indicates

pulsed UV-light treatment helps in preserving the lettuce quality.

In general, UV-light treatment of food may not cause any adverse effect, if

applied in moderate amounts, which is required for microbial inactivation as strongly

suggested by previous studies. However, modification and optimization of the UV-light

treatment might be necessary for successful implementation of the process in some foods.

Water disinfection by UV-light

Water is abundantly used in almost all the processing industries including food

industries. Water may contain several pathogenic microorganisms including protozoa,

bacteria and viruses. UV-light, ozonation, and chlorination are some of the commonly

30

used disinfection methods for inactivation of these microorganisms. Ultraviolet light

does not produce any toxic by-products. UV-light has been used for disinfection of

drinking water since the early 1900s. In addition to the disinfection of drinking water,

UV-light can also be used for disinfection of water used in semiconductor,

pharmaceutical, food or other industries and for sanitation of waste water. Currently low

pressure (typically a monochromatic source at 254 nm) and medium pressure lamps

(typically a polychromatic source with 40 to 50% of energy directly used for disinfection)

commonly used for disinfection of water (Masschelein, 2002), which are continuous UV-

light sources. Pulsed UV-light sources are now gaining attention as the effectiveness of

disinfection can be enhanced several folds. Furthermore, pulsed UV-light provides a

mercury free disinfection system. The UV-light dose required for inactivation of

microorganisms is listed in Table 2.7.

The UV-light treated microorganisms, upon exposure to light in the range of 330

to 480 nm results in photoreactivation (Walker, 1984). The photoreactivated

microorganisms are much more resistant to UV-light and thus require higher dose for

inactivation (Guerrero-Beltran and Barbosa-Canovas, 2004; Hoyer, 1998; Sastry et al.,

2000). As it can be seen from Table 2.7, cells require more energy for inactivation after

being treated once and reactivated. Therefore, higher UV doses are needed for complete

inactivation of pathogenic microorganisms.

31

Table 2.7. Ultraviolet light exposure (at 254 nm) required for 4-log10 reduction of pathogens in drinking water*.

Microorganism Exposure required

without reactivation (J/cm2)

Exposure required with reactivation

(J/cm2) Citrobacter freundii 80 250

Enterobacter cloacae 100 330 Enterocolitica faecium 170 200

Escherichia coli ATCC 11229 100 280 Escherichia coli ATCC 23958 50 200

Klebsiella pneumoniae 110 310 Mycobacerium smegmatis 200 270 Pseudomonas aeruginosa 110 190

Salmonella Typhi 140 190 Salmonella Typhimurium 130 250

Serratia marcescens 130 300 Vibrio cholerae wild isolate 50 210

Yersinia enterocolitica 100 320 * Guerrero-Beltran and Barbosa-Canovas, 2004; Hoyer, 1998; Sastry et al., 2000.

Economics of UV-light disinfection system

Compared to other available disinfection technologies, the cost of UV-light

disinfection systems is competitive or sometimes cheaper. Dunn (1997) estimated that

the treatment cost at 4 J/cm2 with the PureBright® pulsed UV-light treatment system is

0.1¢/ft2 of treated area. The cost includes conservative estimate of electricity,

maintenance, and equipment amortization. Lander (1996) also estimated 0.1¢/ft2 as the

cost of treatment with the PureBright® system, where the estimated cost includes the

electricity, maintenance, and investment in a hooded high intensity lamp and power unit.

Choi and Nielsen (2005) suggested that it will be cost-effective for the apple cider

industry to utilize UV-light pasteurization, because UV-light pasteurizers cost about

$15,000 (Higgins, 2001). Anonymous (1989) reported that the annual power

consumption and lamp replacement costs based on the minimum dosage of 30,000

32

MW.s/cm2 for multilamp and single lamp continuous UV-light disinfection sources as

$2,465 and $3,060 for an 8,000 h run time.

The processing cost for 4 log10 reduction of E. coli in primary waste water by UV-

light, electron beam, and gamma irradiation were, 0.4¢/m3, 1.25¢/m3, and 25¢/m3,

respectively (Taghipour, 2004). This clearly indicates that the UV-light treatment is cost-

effective for inactivation of pathogenic microorganisms.

2.10. Infrared heating

Infrared radiation can be divided into three categories namely near infrared (NIR;

0.75 – 1.4 µm; temperature less than 400oC), mid infrared (MIR; 1.4 – 3 µm; temperature

in the range of 400oC to 1000oC), and far infrared (FIR; 3 – 1000 µm; temperature more

than 1000oC) (Skjoldebrand, 2001; Sakai and Hanzawa, 1994). Shorter wavelength

infrared radiation gives greater penetration depth while giving lesser temperature when

compared to longer wavelength radiation (Skjoldebrand, 2002). Infrared heating has

several advantages such as, 1) higher heat transfer capacity, 2) instant heating because of

direct heat penetration, 3) high energy efficiency, and 4) faster heat treatment, 5) fast

regulation response, 6) better process control, 7) no heating of surrounding air, 8)

equipment compactness, 9) uniform heating, 10) preservation of vitamins, and 11) less

chance of flavor losses from burning of foods (Dagerskog and Osterstrom, 1979; Afzal et

al., 1997; Skjoldebrand, 2002). Far infrared radiation has been used in the industry since

1950s in United States (Skjoldebrand, 2001).

33

Infrared radiation basics

Temperature of the heating element determines the wavelength at which

maximum radiation occurs as described by Planck’s law, Wien’s displacement law, and

Stefan-Boltzman’s law (Sakai and Hanzawa, 1994; Dagerskog and Osterstrom, 1979).

Stefan-Boltzman’s law (Equation 2.3) states that the amount of heat emitted from a black

body (a theoretical object that absorbs 100% of the radiation that fall on it) can be

determined using

4ATQ σ= (Equation 2.3)

where, Q is the rate of heat emission in Js-1, σ is the Stefan-Boltzman’s constant (5.7 x

10-8 Js-1m-2K-4), A is the surface area in m2, and T is the absolute temperature in oK. This

law clearly indicates that even a small increase in temperature can lead to higher rate of

heat emission.

Infrared heating lamp emits radiation in range of wavelengths with varying

intensities. The wavelength at which maximum radiation occurs depends on the lamp

temperature according to Planck’s law (Equation 2.4). As the temperature of the lamp

increases, the wavelength at which maximum radiation occur decreases and the total

energy emitted increases (Figure 2.5).

( )

⎥⎥⎦

⎤

⎢⎢⎣

⎡−

=⎟⎠

⎞⎜⎝

⎛

1

2,

52

2

kTnhc

ob

o

en

hcTE

λ

λ

λ

πλ

(Equation 2.4)

where, E is the emissive power of a black body, h is the Planck’s constant (6.626 x 10-3

34

Figure 2.5. Radiation emissive power spectrum (Modest, 2003).

Js), Co is the speed of light in kms-1, n is the refractive index of the medium, λ is the

wavelength in µm, k is the Boltzmann’s constant (1.3806 x 10-23 JK-1) and T is the

absolute temperature in oK. Figure 2.5 shows the Planck’s curve for black body radiation

at different temperatures.

Wien’s law states that the wavelength corresponding to maximum emission is

inversely proportional to the black body temperature (Equation 2.5).

T2900

max =λ (Equation 2.5)

where, λmax is the wavelength corresponding to maximum emission in µm and T is the

absolute temperature of the black body in oK.

35

Interaction of infrared radiation with food materials

As food is exposed to infrared radiation, it is absorbed, reflected, or scattered

(blackbody does not reflect or scatter). Absorption intensities at different wavelengths by

food components differ. As the infrared radiation is absorbed by food components, the

vibration and rotation of the molecules change because of a decrease or increase in the

distance between atoms, movement of atoms, or vibration of molecules (Skjoldebrand,

2002). A significant portion of the infrared radiation is reflected back depending upon

the wavelength. For instance, at NIR wavelength region (λ<1.25 µm), approximately

50% of the radiation is reflected back, while less than 10% radiation is reflected back at

FIR wavelength region (Skjoldebrand, 2002). Most organic materials reflect 4% of the

total reflection producing shine of polished surfaces. The rest of the reflection is because

of the body reflection where radiation enters the food material and scatters producing

different color and patterns (Dagerskog, 1978).

The rate of heat transfer to food material depends on several factors such as the

composition, temperature of the infrared lamp, moisture content of the food material,

shape and surface characteristics of the food material. A crucial factor in infrared heating

of food is the penetration depth which is defined as the 37% of the unabsorbed radiation

energy. As indicated earlier, penetration depth depends on the wavelength region.

Penetration depth of NIR is approximately ten times higher than the FIR wavelength

region (Skjoldebrand, 2002).

36

Inactivation mechanism of infrared heat treatment

Infrared radiation consists of wavelengths between 0.5 and 1000 μm (Rosenthal,

1992). Far Infrared Radiation (FIR) (3 to 1000 μm) is used in the pasteurization of food

products, because the absorption of infrared energy by water and organic materials is

high in this wavelength region (Sawai et al., 1995). Infrared heating is effective in the

inactivation of bacteria. Though, the effect of FIR irradiation is not clear, several

researchers suggested several possible targets for inactivation such as DNA, RNA,

ribosome, cell envelope, and proteins in the cell (Sawai et al., 1995). The pasteurization

effect of far infrared irradiation is much greater than thermal conduction because of the

absorption of infrared energy within a thin domain near the surface and bulk temperature

of the suspension. This was verified by the fact that inactivation of bacteria was observed

even when the bulk temperature was negligible because of cooling (Sawai et al., 1995).

The sensitivity of E. coli to rifampicin and cholormpenicol increased following heat

treatment with FIR irradiation and thermal conduction, suggesting that the damage to

RNA polymerase and ribosome occurred which leads to cell death (Sawai et al., 1995).

Infrared heat treatment

As the wavelengths of radiation emitted in the NIR and MIR ranges can be

utilized for instantaneous heating within seconds, they have been an interest to the food

industry. NIR has a penetration depth of up to 5 mm for some food products

(Skjoldebrand, 2001). Infrared heating can be utilized for several applications in the food

industry such as 1) drying vegetables, fish, pasta, and rice, 2) heating flour, 3) frying

meat, 4) roasting cereals, coffee, and cocoa, 5) baking pizza, biscuits, and breads, 6)

37

thawing, and 7) surface pasteurization of bread and packaging materials (Skjoldebrand,

2001). Skjoldebrand et al. (1994) reported that baking time was 25-50% shorter for

infrared radiation when compared to a conventional oven for comparable energy

consumption. The weight loss associated with infrared radiation was 10-15% lower than

with an oven, and the quality of the final products from both methods were comparable.

E. coli O157:H7 suspended in 0.05 M physiological phosphate buffered saline

was treated with infrared radiation with a radiative power of 3.22x10-1 J/s-cm2 on the

surface of bacterial suspension (Sawai et al., 1995). The temperature of the bacterial

suspension increased exponentially from approximately 280oK to 336oK during a 10 min

infrared irradiation treatment. After pasteurization by FIR, the cell suspension was

diluted with physiological saline solution and plated on the agar surface. Two types of

agar plates were used: selective and non-selective. Sensitivity disk agar-N containing

penicillin G (PCG), chloramphenicol (CP), nalidixic acid (NA), and riampicin (RFP)

were used as a selective agar, and sensitivity disk agar-N was used as a non-selective

agar. Selective agar with the reagents was used in order to characterize the type of injury

of E. coli O157:H7. PCG, CP, NA, and RFP inhibited the synthesis of cell wall, protein,

DNA, and RNA, respectively. After infrared irradiation, E. coli O157:H7 becomes

sensitive to RFP and CP, which may be because of the damage of RNA polymerase and

ribosome in E. coli by infrared radiation. Approximately, 3 log10 reduction of E. coli was

obtained after an 8 min treatment, when chloramphenicol selective agent was used. The

validity of this result was verified by the comparison of the effect with UV-light

treatment. Approximately, a 1 log10 reduction of E. coli was obtained after 20 s when a

chloramphenicol selective agent was used. The authors concluded that the FIR

38

pasteurization effect was because of a heating effect after comparison with conventional

convective heating.

The efficacy of infrared heating on microbial reduction of milk was investigated

by Giraffa and Bossi (1984). They reported that infrared heat treatment of milk at 65oC

and 1000 L/h flow rate resulted in reductions of standard plate count, Psychotropic

bacteria, Coliforms, Enterococci, Lactic acid bacteria (grown in MRS agar), Lactic acid

bacteria (grown in M17 agar), and spores by 63%, 96% 99.9%, 80%, 71%, 60%, and

21%, respectively.

Rosenthal et al. (1996) investigated the surface pasteurization effect of infrared

radiation in cottage cheese. Surface heating of the cheese was done by Philips infrared

spotlights (250 J/s) at a distance of 2.5 to 3 cm from the cheese surface. After the

treatment, cheeses were examined for yeast, mold, and coliform counts during 8 weeks of

storage at 4oC. The initial yeast and mold counts before infrared pasteurization were <10

cells/g. Even after 8 weeks storage at 4oC, less than 100 yeast and mold cells/g were

found, suggesting that infrared pasteurization was effective for surface sterilization. The

corresponding log10 reductions of yeasts and molds were approximately 3. However, the

number of yeast and mold cells was reduced to only approximately 1 log10 CFU/g at a 1

cm depth from the cheese surface.

Hashimoto et al. (1992a) investigated the effect of far-infrared irradiation on

pasteurization of bacteria suspended in liquid medium below lethal temperature. E. coli

745 and S. aureus 9779 cultures were suspended in 0.05 M phosphate buffer (pH=7.0)

and dispensed in a stainless steel Petri dish with a thermally insulated side walls. The

Petri dish was covered with aluminum foil to reflect infrared radiation. The Petri dish

39

was placed on a temperature controlled plate, where the temperature was maintained

from 263 to 283oK by a coolant, and the plate was on a reciprocating shaker. The

infrared irradiation apparatus was kept at a 15 cm distance from the bacterial suspension,

and the source temperature was from 773 to 943oK. The bacterial suspension was

infrared treated under agitation (180 rpm) and cooled rapidly using a plate kept at 278 oK

immediately after the treatment. A reduction of more than 4.5 log10 CFU/ml of S. aureus

was achieved with an irradiation power of approximately 7.5x10-7 J/s.cm2 when using

selective agar (standard method agar enriched with 8% sodium chloride); whereas, a

reduction of approximately 3.5 log10 CFU/ml was achieved using non-selective agar

(standard method agar). Similarly, a log10 reduction of approximately 2 log10 CFU/ml

and approximately 0.5 log10 CFU/ml was obtained for E. coli 745 and S. aureus 9779,

respectively, when selective agar (Nutrient agar enriched with 0.06% sodium

deoxycolate) and non-selective agar (Nutrient agar) were used.

The FIR effect on the pasteurization of bacteria on or within wet-solid medium

was evaluated by Hasimoto et al. (1992b). E. coli 745 and S. aureus 9779 culture were

surface plated on nutrient agar and standard method agar, respectively. After surface

plating, the bacteria were covered with agar medium to a thickness between 1 to 5 mm.

An FIR heater with a reflector which irradiated 2.46x103 to 7.01x10-1 J/s.cm2 power on

the agar plate was used in the experiment. A complete inactivation of E. coli was

obtained with irradiation at 4.36x10-1 J/s-cm2 for 6 min., which corresponds to

approximately 2 log10 CFU/plate when there is no medium added. However, when the

medium thickness was 1 mm and 2 mm, approximately, 1 log10 CFU/plate was obtained

under the same experimental condition.

40

Hashimoto et al. (1993) also investigated the irradiation power effect on IR

pasteurization below reaching the lethal temperature of bacteria. E. coli 745 and S.

aureus 9779 culture were suspended in 0.05 M phosphate buffer (pH=7.0). FIR and Near

Infrared Radiation (NIR) were used in the experiment. The irradiation power for the NIR

heater was calculated using surface temperature and emissivity of the heater. The

irradiation power of FIR was calculated using a Fourier transform infrared

spectrophotometer. Approximately 4 log10 CFU/ml and 1 log10 CFU/ml reductions of S.

aureus were obtained by FIR and NIR, respectively, when the irradiation power was

7.57x 10-1 J/s-cm2, which indicates that the FIR heating is more effective in inactivating

the microbial population than NIR heating.

Jun and Irudayaraj (2003) studied inactivation of Aspergillus niger inoculated on

corn meal by infrared radiation with or without a bandpass filter (5.45 to 12.23 μm). A

reduction of approximately 2 log10CFU/g was obtained with and without the filter after a

5 min treatment time; however, the log10 reduction obtained with a filter was higher than

the log10 reduction obtained without a filter. Similarly, Fusarium proliferatum was

inoculated on corn meal and treated with infrared radiation with or without a bandpass

filter and resulted in approximately 1.5 and 2 log10 CFU/g reductions, respectively, with a

5 min treatment time.

41

2.11. Determination of mode of microbial inactivation

It is necessary to understand the basic inactivation mechanism of an emerging

technology in order to optimize the system for better inactivation of pathogenic

microorganisms. Selected examples are discussed below.

Transmission electron microscopy

Transmission electron microscopy (TEM) can be used effectively for

investigation of the mode of bacterial inactivation, as these microscopes can help in

viewing cellular level details of microorganisms. A TEM is a modern, sophisticated

microscope, which uses an electron beam to observe internal structures of

microorganisms. The resolution of TEM is 1,000 times better than the light microscope

enabling it to distinguish points closer than 0.5 nm (Prescott et al., 1999). As the

electrons are absorbed and scattered by microorganisms very easily, very thin slices in

the order of 20 to 100 nm need to be used. The bacterial cell is treated with various

chemicals to stabilize the cell structure, cut into a thin slice using a diamond or glass

knife, stained to increase the contrast, and exposed to an electron beam. As the

composition of the cell components differ, the intensity of electron scattering also varies

and thus produces an image of the internal structure of the bacteria. Several researchers

have used the electron microscopy techniques successfully for the investigation of

inactivation mechanisms of several novel technologies such as pulsed electric field,

antimicrobial agents, etc. (Brouillette et al., 2004; Calderon-Miranda et al., 1999; Liu et

al., 2004). The effect of pulsed electric field and nisin on Listeria innocua in skim milk

was investigated using TEM by Calderon-Miranda et al. (1999). They noticed several

42

futures of pulsed electric field damaged cell such as lack of cytoplasm, cell wall damage,

cytoplasmic clumping, increase in cell membrane thickness, poration of cell wall and

leaching of cellular content. The ruptures of cell wall and cell membrane were observed

at selected electric field intensities. Liu et al. (2004) explained that the inactivation of

bacteria by chitosan is because of cell wall damage by using TEM. The outer membrane

of chitosan treated E. coli was altered after the chitosan treatment. The cell membrane of

chitosan-treated Staphylococcus aureus was disrupted and the cellular contents were

leaked.

Fourier transform infrared spectroscopy

Infrared spectroscopy is a chemical analytical method which can be used to

determine the chemical and structural information of the target material based on

vibration transitions. Various food/microbial components absorb infrared light at specific

wavelengths. Thus, infrared spectroscopy produces a fingerprint of spectral absorption

characteristics of the biological components by providing absorption/transmission

characteristics over time. The spectrum obtained is transformed from the time domain

into frequency domain by Fourier transformation, so that absorption with respect to a

particular wavelength could be assessed.

Fourier Transform Infrared Spectroscopy (FTIR) can be used to discriminate

pathogenic microorganisms by spatially resolving the structural and compositional

information of the microorganisms at molecular level (Yu and Irudayaraj, 2005). Liu et

al. (2004) utilized FTIR for determination of the interaction between chitosan and a

synthetic phospholipids membrane in an effort to understand the basic inactivation

43

mechanism. Several researchers successfully utilized FTIR for the discrimination of

microorganisms and to investigate the changes in chemical composition because of

several processes. Therefore, the use of TEM and FTIR will be beneficial for the

investigation of inactivation mechanisms.

2.12. Summary of literature review

Contamination of pathogens in food and water is a serious threat to the industry

from a health and economic perspective. Staphylococcus aurues food poisoning alone

causes 185,060 illnesses, 1,753 hospitalizations, and 2 deaths in United States annually

(Mead et al., 1999) estimated to cost about $1.2 billion (Buzby et al., 1996). Several

novel disinfection methods are utilized by the industry to inactivate the pathogens

effectively; however, the challenge posed in the preservation of food quality limits their

application. Therefore, there is always a need to optimize the existing processing method

and/or identify a new method for inactivation of pathogenic microorganisms while

preserving the quality of the food.

UV-light has been used as a bactericidal agent for over a century, because it is

effective for inactivation of pathogens on a surface and in clear liquid. However, it has a

poor penetration capacity and hence was not utilized for treatment of opaque solutions.

Pulsed UV-light is the application of broadband UV-light in a pulsed mode, wherein the

instantaneous intensity of the UV-light is increased significantly. Increased UV-light

intensity and the shocking effect of pulses may aid in enhancing the effectiveness of UV-

light on microbial inactivation. Optimization of pulsed UV-light and a proper equipment

design may result in a disinfection process for opaque food products such as milk. Pulsed

44

UV-light may provide a cheaper alternative to existing pasteurization methods as the

capital and operational costs are comparatively less than existing technologies.

Furthermore, pulsed UV-light treatment also fortifies the Vitamin D content of the food

being treated.

Infrared heating is a cost effective method of heat treatment which has several

advantages such as high heat transfer rate and energy savings. Infrared heating can also

be utilized to heat selectively a particular food component or target of interest such as

bacterial cells without heating other components. Therefore, selective heating may result

in better product quality as only the bacterial cells are heated. Though not widely utilized

for bacterial inactivation, previous studies indicate that far infrared heating is very

effective on bacterial inactivation. Though, the energy consumption is almost 50% less

than the regular heating process, far infrared heating also results in better product quality.

Therefore, successful application of infrared heat treatment will result in cost reduction

and better quality food products.

FTIR and TEM can be successfully used for the investigation of inactivation

mechanisms of microorganisms. Several researchers have used these techniques to

identify the cause of microbial inactivation for several emerging inactivation

technologies. Identification of inactivation of mechanism of pulsed UV-light and

infrared heating will result in better understanding of the underlying process, which in

turn results in better process optimization.

As the literature review substantiates, there was not much research work done in

optimization of pulsed UV-light and infrared heating inactivation of S. aureus in milk. In

this study, S. aureus culture inoculated in milk or milk foam was treated with pulsed UV-

45

light and infrared heating and the degree of S. aureus inactivation were determined.

Furthermore, the inactivation mechanism of S. aureus using pulsed UV-light was

investigated using spectroscopic and microscopic studies. The efficacy of the pulsed

UV-light for inactivation of resistant B. subtilis spores was also investigated to determine

the applicability of UV-light for inactivation of spores.

46

3. Inactivation of Staphylococcus aureus by Pulsed

UV-Light Sterilization*

3.1. Abstract

Pulsed UV-light is a novel technology to inactivate pathogenic and spoilage

microorganisms in a short time. Efficacy of pulsed UV-light for the inactivation of

Staphylococcus aureus as suspended or agar seeded cells was investigated. A 12, 24, or

48 ml cell suspension in buffer was treated under pulsed UV-light for up to 30 s and a 0.1

ml of sample was surface plated on Baird-Parker agar and incubated at 37oC for 24 h to

determine log10 reductions. Also, a 0.1 ml of cell suspension in peptone water was

surface plated on Baird-Parker agar plates and the plates were treated under pulsed UV-

light for up to 30 s. The treated and untreated plates were incubated as before. A 7 to 8

log10 CFU/ml reduction was observed for suspended and agar seed cells treated for 5 s or

higher treatment times. In the case of suspended cells, the sample depth, time, treatment,

and interaction were significant (p<0.05). In case of agar seeded cells, the treatment time

was significant (p<0.05). This study clearly indicates that pulsed UV technology has

potential for inactivation of pathogenic microorganisms.

* This article was originally published in Journal of Food Protection. Reprinted with permission from the Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, Iowa, USA. The original citation is as follows: Krishnamurthy, K1., A. Demirci1, and J. Irudayaraj2. 2004. Inactivation of Staphylococcus aureus by pulsed UV light treatment. J. Food Prot. 67:1027-1030. 1Department of Agricultural and Biological Engineering, Pennsylvania State University, University Park, PA, USA, 2Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, USA. Only portions of the article relevant to the thesis are presented and the content was formatted as per thesis office requirements.

47

3.2. Introduction

Foodborne diseases are estimated to cause approximately 76 million illnesses,

325,000 hospitalizations, and 5,000 deaths annually in the United States (Mead et al.,

1999). Therefore, foods contaminated with pathogenic microorganisms, such as,

Salmonella spp., Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni,

Escherichia coli O157:H7, and Listeria monocytogenes remain a major public health

concern. Among these pathogens, S. aureus is one of the common causes of disease

(Martin et al., 2001). According to Centers for Disease Control and Prevention (CDC),

there have been 17,248 and 1,413 cases of S. aureus cases reported during 1973-87 and

1993-97, respectively, accounting for 14% and 1.6% of all cases because of bacterial

pathogens (Bean and Griffin, 1990; Olsen et al., 2000).

Several technologies have been evaluated for the inactivation of these pathogens,

such as, heat treatment, cold temperature treatment, irradiation, microwave radiation,

pulsed electric field, magnetic field, high pressure, and ohmic heating treatments (Juneja

and Sofos, 2002). In this study, effect of pulsed UV-light treatment on inactivation of a

pathogen, S. aureus, was investigated. Though, UV-light treatment is effective in

inactivation of pathogens, it may have several disadvantages such as oxidation of

unsaturated fats, poor penetration capability of UV-light, and destruction of some

vitamins. However, these may be reduced or eliminated by shorter treatment time and

thin layer treatment.

UV-light has been used as a bactericidal agent since as early as 1928 (Xenon,

2003). UV-light is a portion of the electromagnetic spectrum ranging from 100 to 400

nm wavelengths. UV-light in the wavelength range of 100 to 280 nm has germicidal

48

implications (EPA, 1999). UV-light sterilization provides a cost effective alternative to

the existing heat pasteurization techniques and preservation methods. Also, taste

degradation of the food material subjected to UV-light treatment is minimal, since UV-

light treatment can be accomplished at an ambient temperature (Hollingsworth, 2001).

UV-light damages the DNA by forming pyrimidine dimers (McDonald et al., 2000). This

dimer prevents the microorganism from DNA transcription and replication, which leads

to cell death. Several researchers demonstrated that UV-light can be used for the

inactivation of pathogens without adversely affecting the quality of food (Allende and

Artes, 2003; Rowan et al., 1999; Smith et al., 2002; Yaun et al., 2003).

The UV-light treatment can be accomplished in two modes namely continuous

UV-light or Pulsed UV-light. Continuous UV-light has several disadvantages such as

poor penetration depth and low dissipation power, whereas pulsed UV-light sterilization

has higher penetration depth and dissipation power. Pulsed UV-light treatment is more

effective and rapid for microorganism inactivation compared to continuous UV-light

sources, since the energy is multiplied many fold (CFSAN-FDA, 2000; Dunn et al.,

1995). Power dissipation from a continuous UV-light system ranges from 100 to 1000 W

(Demirci, 2002); however, a pulsed UV-light system can produce peak power distribution

as high as 35 MW (McDonald et al., 2002; McGregor et al., 1997). In pulsed UV-light

treatment, the energy is stored in a high power capacitor and released constantly in a

short period of time (nano to microseconds). This produces several high energy flashes

per second and hence the microorganisms are inactivated effectively because of high UV-

content during the flash and constant disturbance caused by pulses. Moreover, the pulsed

UV-light provides cooling periods between pulses and hence reduces the temperature

49

build-up as compared to continuous UV-light because of short pulse duration and cooling

period between pulses. McDonald et al. (2000) compared the effectiveness of a

continuous UV light source and a pulsed UV-light source for the decontamination of

surfaces. The authors reported that the almost identical level of inactivation of Bacillus

subtillis was obtained with an energy level of 4 x 10-3 J/cm2 of pulsed UV-light source

instead of 8 x 10-3 J/cm2 continuous UV-light source.

Chang et al. (1985) investigated the effect of continuous UV light on inactivation

of E. coli, S. Typhi, Shigella sonnei, Streptococcus faecalis, and S. aureus. E. coli, S.

aureus, S. sonnei, and S. Tyhi exhibited similar resistances to the UV-light and required

approximately 7 x 10-3 J/cm2 energy to obtain 3 log10 reduction. However, the resistance

exhibited by S. faecalis was higher and required 1.4 times higher dose than the above

mentioned microorganism to get 3 log10 reduction.

On the other hand, a 6 log10 reduction of L. monocytogenes, E. coli, S. Enteritidis,

Psudeomonas aeruginosa, B. cerus, and S. aureus seeded on agar surface was obtained

after a 20 μs treatment with pulsed UV-light (Rowan et al., 1999). Sonenshein (2003)

investigated the effect of high-intensity pulsed UV light with an energy level of 15.8

J/cm2.s on inactivation of B. subtillis spores. A complete inactivation (7 to 8 log10

reduction) was obtained with 1 s exposure of UV-light when the samples were placed at

the lamp axis and at the midpoint of the lamp. The effectiveness of pulsed UV-light for

the inactivation of E. coli O157:H7 on inoculated alfalfa seeds was demonstrated by

Sharma and Demirci (2003). A complete inactivation of E. coli O157:H7 (4.80 log10

CFU/g) was obtained after 30 s treatment when the thickness of alfalfa seeds was kept at

1.02 mm.

50

Stermer et al. (1987) investigated the effect of pulsed UV radiation on

inactivation of bacteria in lamb meat. With 4 x 10-3 J/cm2 energy, 99.9% of the naturally

occurring flora of lamb (mostly Pseudomonas, Micrococcus, and Staphylococcus species)

was inactivated. The bactericidal effectiveness of pulsed UV-light was investigated by

Bank et al. (1990). Bacterial monolayers of S. epideridis, Pseudomonas aeruginosa, E.

coli, S. aureus, or Serratia marcescens on Trypticase soy agar plates were exposed to

pulsed UV-light. A 60 s treatment time at 31 cm distance from the light source resulted

in 6 to 7 log10 reduction of viable bacterial population (~4 x 10-4 J/cm2).

Though, Bank et al. (1990) and Rowan et al. (1999) investigated the effect of

pulsed UV-light on inactivation of S. aureus on solid surface, there is a need to

investigate the effect of pulsed UV-light on liquid medium, which represents liquid food

systems such as milk. In addition to liquid medium, the effect of pulsed UV-light on

solid agar surface was also investigated as a comparative method. The overall purpose of

this study was to investigate the possibility of using pulsed UV-light as an alternative

method for the inactivation of S. aureus and to investigate the effect of various

parameters such as depth of sample, time of exposure, and medium of introduction (solid

[agar seeded cells] or liquid [suspended cells]).

51

3.3. Materials and Methods

Microorganism

Staphylococcus aureus (ATCC 13311) was obtained from the Penn State Food

Microbiology Culture Collection. Cells were grown in 150 ml of Tryptic Soy Broth

(TSB) (Difco, Sparks, MD) at 37oC for 24 h, and harvested by centrifugation (Sorvall

STH750, Kendro Lab Products, Newtown, CT) at 3,300 x g for 25 minutes at 4oC.

Sample preparation

The cells to be treated by pulsed UV-light were prepared as suspension cells and

agar seeded cells, which represent liquid and solid food systems.

i) Suspended Cell: After centrifugation, the pellet was resuspended in 100 ml of sterile

0.1 M % phosphate buffer (pH=7.0) to obtain viable cell populations of approximately 7

to 8 log10 CFU/ml of S. aureus. A 12, 24, or 48 ml cell suspension in buffer was

transferred to sterile aluminum containers with a diameter of 7 cm (VWR International,

Buffalo Grove, IL).

ii) Agar seeded cell: The pellet was resuspended in 100 ml of sterile 0.1% peptone

water. The resulting inoculum solution had approximately 7 to 8 log10 CFU/ml of S.

aureus and the cell suspension was serially diluted up to 10-5 dilution. A 0.1 ml of cell

suspension from each dilution and inoculum was surface plated on Baird-Parker agar

plates. To facilitate direct counting of treated and untreated plates, regular plastic Petri

dishes were used rather than aluminum containers.

52

Pulsed UV-light treatment

Pulsed UV-light treatment was carried out with a lab scale, batch pulsed light

sterilization system (SteriPulse®-XL 3000, Xenon Corporation, Woburn, MA). The

system generated 1.27 J/cm2 per pulse at 1.8 cm below the quartz window for an input

voltage of 3,800 V and with 3 pulses per second as per the manufacturer. The distance

between the quartz window and the centre axis of the UV-light strobe was 5.8 cm. The

output from the pulsed UV-light system followed a sinusoidal wave pattern with 1.27

J/cm2 per pulse being the peak value of the pulse. Power values of UV-light treatments

used in this manuscript are based on peak value of the pulse (1.27 J/cm2 per pulse). The

pulse width (duration of pulse) was 360 µs. The suspended cell samples with varying

volumes were treated under pulsed UV-light for 1, 2, 3, 4, 5, 10, 15, or 30 s at a distance

of 8 cm from the quartz window. Similarly, agar seeded plates are treated under pulsed

UV-light for 1, 2, 3, 4, 5, 10, 15, or 30 s at a distance of 8 cm from the quartz window.

During the pulsed UV-light treatment, the temperature of agar plates and the suspended

cell liquid were monitored using a type K thermocouple (Omegaette HH306, Omega

Engineering Inc., Stamford, CT).

Microbiological analysis

i) Suspended cell: Untreated samples and samples immediately after pulsed UV-light

treatment were analyzed for surviving populations of S. aureus. A 1 ml sample from the

sample cup was serially diluted with 0.1 M phosphate buffer. This is followed by

surface-plating of a 0.1 ml sample on Baird-Parker agar. After incubating at 37oC for 24

53

h, the colonies were enumerated. The log10 reduction was calculated by subtracting the

treatment and control plate counts. Each experiment was replicated three times.

ii) Agar seeded cell: Pulsed UV-light treated agar plates were incubated for 24 h at

37oC. The higher dilution plates with no colonies were discarded and the colonies in the

low dilution plates were counted and reported. Three replications for each experiment

were performed.

Statistical analysis

Statistical significant differences between treated and untreated cells were tested

using the General Linear Model of ANOVA with 2-way interaction with MINITAB

software (version 13.3, Minitab Inc., State College, PA). A 95% confidence interval was

used.

3.4. Results and Discussion

Suspended cell treatment

In order to demonstrate effectiveness of pulsed UV-light on liquid medium, S.

aureus cells were suspended in 0.1 M phosphate buffer and treated by pulsed UV-light up

to 30 s at a distance of 8 cm from UV strobe. A complete inactivation was obtained for

samples treated with 5 s for all sample sizes. The average corresponding log10 reduction

was 7.50 log10 CFU/ml (Figure 3.1).

54

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0 5 10 15 20 25 30Time (sec)

log 1

0 CFU

/ml r

educ

tion

12 ml

24 ml

48 ml

Figure 3.1. Log10 reductions of Staphylococcus aureus after pulsed UV-light sterilization in suspended cell.

The pulsed UV-light was very effective in inactivating S. aureus in suspension.

Complete inactivation was also observed for 1 and 2 s treatments of 12 and 24-ml

samples, respectively. However, after 1-s treatment, a 4.6 log10 and 1.5 log10 reductions

were obtained in 24 and 48 ml samples, respectively. For the 48 ml sample, the log10

reduction increased exponentially in 5 s. As expected, the log10 reduction increased with

treatment time because of increase in number of pulses. The effect of the sample depth

was significant (p<0.05). The temperature of the sample increases, as the treatment time

increases after several seconds (Figure 3.2), however, no significant temperature increase

was observed during the first 5 s. Pulsed UV-light treatment is considered non-thermal,

but holds only for short time treatments. Temperature increases as absorbed energy

accumulates during longer treatments. During a 20-s treatment time, the temperature

55

increase was about 20oC for a 12 ml phosphate buffer sample. Finally, statistical

analysis suggested that the effect of treatment time and the interaction (treatment

time*depth) were significant (p<0.05).

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20Time (sec)

Tem

pera

ture

(o C)

Phosphate buffer - 12 ml

Baird parker agar

Figure 3.2. Temperature profile of phosphate buffer and Baird-Parker agar base during pulsed UV-light treatment.

Agar seeded cells

In order to demonstrate the effectiveness of pulsed UV-light on solid surfaces,

agar seeded S. aureus cells were treated by pulsed UV-light for up to 30 s at a distance of

8 cm from the UV strobe. The corresponding power available at 8 cm distance away

from the quartz window were 0.99, 1.98, 4.95, and 29.7 J/cm2/s for 1, 2, 5, and 30 s

treatment time, respectively. A 5-s treatment inactivated all S. aureus to yield about 8.5

log10 reduction (Figure 3.3) contributing to an effective inactivation of S. aureus (P<0.05)

56

on the agar surface, when the distance between agar seeded cells and UV strobe was 8

cm. Bank et al. (1990) obtained 6 to 7 log10 reduction of agar seeded cells after 60 s

treatment, when the distance from UV strobe was 31 cm. The corresponding power

obtained by the sample is 6.67x10-6 J/cm2/s. Since, the distance of quartz window from

the sample for our system was different from Bank et al. (1990), comparing the log10

reduction from both the cases will be futile. A 5 log10 reduction was obtained after a 1-s

of treatment. As expected, the log10 reduction of S. aureus increased exponentially with

the treatment time similar to suspended cell solution and complete inactivation was

achieved within 5 s.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 5 10 15 20 25 30Time (sec)

Log 1

0 CFU

/ml r

educ

tion

Figure 3.3. Log10 reductions of Staphylococcus aureus after pulsed UV-light sterilization in seeded agar plates.

The temperature of the agar increased for longer treatment times (Figure 3.2).

However, there is no significant increase in the temperature during the first 5 s and the

inactivation was hypothesized to occur primarily because of the pulsed UV-light and not

57

because of the synergistic effect from the temperature increase. Experiments indicated

that a few colonies survived along the edge of the plate possibly because of the

obstruction of light by the elevated edges of the plate. The intensity of UV-light reduced

with the radial distance from the central axis of the lamp.

Pulsed UV-light treatment is more effective on surface sterilization than

sterilization of liquid medium. Log10 reductions of 5.0 and 1.35 CFU/ml were obtained

for agar seeded cells and suspended cells (when sample volume was 48 ml), respectively

after a 1 s treatment. However, as expected when the depth of the suspended cell was

kept to minimum by having smaller suspended cell volume, suspended cell inactivation

yielded similar results as agar seeded cells. As the sample depth of suspended cell

increases, inactivation level of S. aureus decreases, because of poor penetration capacity

of pulsed UV-light. Therefore, one can increase the effectiveness of pulsed UV-light on

inactivation of suspended cells by minimizing the sample depth and/or increasing the

treatment time.

3.5. Conclusions

The present study clearly shows the potential of pulsed UV-light for S. aureus

inactivation in liquid and solid systems. Complete inactivation of S. aureus can be

achieved within seconds with pulsed UV-light. Therefore, pulsed UV-light can be used

as an alternative to thermal sterilization processes. However, there is a need for

optimization of the experimental parameters to achieve the target inactivation level for

specific applications. Further research is in progress to evaluate the applicability of

pulsed UV-light for the sterilization of various food products.

58

3.6. References

Allende, A., and F. Artes. 2003. UV-C radiation as a novel technique for keeping quality

of fresh processed ‘Lollo Rosso’ lettuce. Food Res. Int. 36:739-746.

Bank, H.L., J.L. Schmehl, and R.J. Dratch. 1990. Bactericidal effectiveness of

modulated UV light. Appl. Environ. Microbiol. 56:3888-3889.

Bean, N.H. and P.M. Griffin.1990. Food-borne disease outbreaks in the United States,

1973-1987: Pathogens, vehicles, and trends. J. Food Prot. 53:804-817.

CFSAN-FDA. 2000. Kinetics of microbial inactivation for alternative food processing

technologies: Pulsed light technology. Atlanta, GA: Center for Food Safety and

Applied Nutrition – Food and Drug Administration. Available at:

http://vm.cfsan.fda.gov/ ~comm/ ift-puls.html. Accessed 19 April 2006.

Chang, J.C.H., S.F. Ossoff, D.C. Lobe, M.H. Dorfman, C.M. Dumais, R.G. Qualls, and

J.D. Johnson. 1985. UV inactivation of pathogenic and indicator microorganisms.

Appl. Environ. Microbiol. 49:1361-1365.

Demirci, A. 2002. Novel processing technologies for food safety. J. Assoc. Food and

Drug Officials. 66(4):1-8.

Dunn, J., T. Ott, and W. Clark. 1995. Pulsed light treatment of food and packaging. Food

Technol. 49:95-98.

EPA. 1999. Ultraviolet radiation. In Alternative Disinfectants and Oxidants Guidance

Manual. Washington, DC: Environmental Protection Agency. Available at:

http://www.epa.gov/safewater/mdbp/pdf/alter/chapt_8.pdf. Accessed 19 April

2006.

Hollingsworth, P. 2001. Niche players pushing UV light applications. Food Technol.

55(12):16-16.

Juneja, V.K., and J.N. Sofos. 2002. Control of foodborne microorganisms. New York,

NY: Marcel Dekker, Inc.

Martin, S.E., E.R. Myers, and J.J. Iandola. 2001. Staphylococcus aureus. In Foodborne

Disease Handbook, 345-382. Hui, Y.H., M.D. Pierson, and J.R. Gorham, ed. New

York, NY: Marcel Dekker Inc.

59

http://vm.cfsan.fda.gov/%20%7Ecomm/%20ift-puls.html

http://www.epa.gov/safewater/mdbp/pdf/alter/chapt_8.pdf

McDonald, K.F., R.D. Curry, T.E. Clevenger, K. Unklesbay, A. Eisenstrack, J. Golden,

and R.D. Morgan. 2000. A comparison of pulsed and continuous ultraviolet light

sources for the decontamination of surfaces. IEEE Trans. Plasma Sci. 28:1581-

1587.

MacGregor, S.J., N.J. Rowan, L. Mcllvaney, J.G. Anderson, R.A. Fouracre, and O.

Farish. 1997. Light inactivation of food-related pathogenic bacteria using a pulsed

power source. Lett. Appl. Microbiol. 27:67-70.

Mead, P.S., L. Slutsker, and V. Dietz. 1999. Food related illness and death in the United

States. Emerg. Infect. Dis. 5:607-625.

Olsen, S.J., L.C. MacKinon, J.S. Goulding, N.H. Bean, and L. Slutsker. 2000.

Surveillance for foodborne disease outbreaks – United States, 1992-1997. Morb.

Mortal. Wkly. Rep. 49(SS-1):1-66.

Rowan, N.J., S.J. MacGregor, J.G. Anderson, R.A. Fouracre, L. McIlvaney, and O.

Farish. 1999. Pulsed-light inactivation of food-related microorganisms. Appl.

Environ. Microbiol. 65:1312-1315.

Sharma, R.R. and A. Demirci. 2003. Inactivation of Escherichia coli O157:H7 on

inoculated alfalfa seeds with pulsed ultraviolet light and response surface

modeling. J. Food Sci. 68(4):1448-1453.

Smith, W.L., M.C. Lagunas-Solar, and J.S. Cullor. 2002. Use of pulsed ultraviolet laser

light for the cold pasteurization of bovine milk. J. Food Prot. 65(9):1480-1482.

Sonenshein, A.L. 2003. Killing of Bacillus spores by high-intensity Ultravaviolet light. In

Sterilization and Decontamination Using High Energy Light. Woburn, MA:

Xenon Corporation.

Stermer, R.A., M. Lasater-Smith, and C.F. Brasington. 1987. Ultraviolet radiation – an

effective bactericide for fresh meat. J. Food Prot. 50(2):108-111.

Xenon. 2003. Sterilization and Decontamination using high energy light. Woburn, MA:

Xenon Corporation.

Yaun, B.R., S.S. Sumner, J.D. Eifert, and J.E. Marcy. 2003. Response of Salmonella and

Escherichia coli O157:H7 to UV energy. J. Food Prot. 66:1071-1073.

60

4. Inactivation of Staphylococcus aureus in Milk and

Milk Foam by Pulsed UV-light Treatment and Surface

Response Modeling

4.1. Abstract

Pulsed UV-light is a novel technology that can be used to inactivate pathogens in

a short time. In this study, efficacy of pulsed UV-light was investigated for inactivation

of S. aureus in milk and milk foam, as it is a pathogen of concern in milk and milk

products. A 12, 30, or 48-mL of cell suspension in milk was treated under pulsed UV-

light for 30, 105, or 180 s. The reduction obtained varied from 0.16 to 8.55 log10 CFU/ml

demonstrating the ability of pulsed UV-light to inactivate S. aureus. Complete

inactivation was obtained at (1) 8-cm sample distance from quartz window, 30-ml sample

volume, and 180-s time combination and (2) 10.5-cm sample distance from quartz

window, 12-ml sample volume, and 180-s treatment time combination. In case of milk

foam, reductions up to 6.61±0.11 log10 CFU/g were obtained at several conditions. It was

noted that there was a significant increase in the milk temperature during pulsed UV-light

treatment, which might have also contributed to microbial inactivation.

61

4.2. Introduction

In 2000, the United States produced 76 million tons of milk worth more than $20

billion (IDFA, 2001). Milk is used in the manufacture of dairy products as well in the

manufacture of a wide variety of food products (IDFA, 2001). Milk may contain

spoilage and pathogenic microorganisms because of improper handling and

contamination from both inside and outside of udder (Bramley and McKinnon, 1990).

Conventionally, thermal pasteurization is used to inactivate microorganisms in the milk.

However, thermal pasteurization has several disadvantages such as high energy demand

and high capital and operational cost. In addition, during thermal pasteurization, warm

areas in the heat exchangers are conducive for the growth of Staphylococcus aureus, a

pathogenic microorganism, which produces heat-resistant toxins.


325,000 hospitalizations, and 5,200 deaths annually in the United States (Crutchfield and

Roberts, 2000). Therefore, foods contaminated with pathogenic microorganisms, such as

Salmonella spp., Clostridium perfringens, S. aureus, Campylobacter jejuni, Escherichia

coli O157:H7, and Listeria. monocytogenes remain a major public health concern.

Among these pathogens, S. aureus is the most common cause of suppurating infections

(Martin et al., 2001). Based on the data from the Centers for Disease Control and

Prevention (CDC), 185,060 illnesses, 1,753 hospitalizations, and 2 deaths occur annually

because of staphylococcal food poisoning, respectively (Mead et al., 1999). S. aureus

infections and intoxications from food sources are estimated to cost about 1.2 million

dollar annually (Buzby et al., 1996). Milk and milk products often associate with S.

62

aureus contamination. Therefore, it is necessary to inactivate the pathogens including S.

aureus in milk more effectively in order to ensure its safety.

Although there are several technologies available for inactivation of S. aureus and

other pathogens, they may change the color, flavor, and/or texture of food products.

Therefore, there is a need for novel processes that can eliminate pathogens while

maintaining the quality of milk. Pulsed UV-light treatment is a potential microbial

inactivation method. The bactericidal effect of UV-light was known as early as 1892

(Kime, 1980). UV-light is a portion of electromagnetic spectrum ranging from 100 to

400 nm wavelengths and provides a cost-effective alternative to existing heat

pasteurization techniques and preservation methods. Also, taste degradation of the milk

subjected to UV-light treatment can be minimal, since UV-light treatment can be

accomplished at ambient temperature (Hollingsworth, 2001). UV-light damages the

DNA by forming thymine dimers (Bank et al., 1990; Miller et al., 1999). These dimers

prevent the microorganism from accomplishing DNA transcription and replication and

thereby lead to cell death (Miller et al., 1999).

The UV-light sterilization can be accomplished in two modes: continuous UV-

light or pulsed UV-light. Pulsed UV-light is a more effective and rapid way of

inactivating microorganisms than continuous UV-light, since the instantaneous power is

multiplied many fold in case of pulsed UV-light (CFSAN-FDA, 2000; Dunn et al., 1995).

McDonald et al. (2000) compared the effectiveness of continuous UV-light and the pulsed

UV-light sources for surface decontamination. The authors reported that similar levels of

inactivation of Bacillus subtilis were obtained with 4 mJ/cm2 of pulsed UV-light and 8

mJ/cm2 continuous UV-light.

63

Chang et al. (1985) investigated the effect of continuous UV light for the

inactivation of E. coli, Salmonella Typhi, Shigella sonnei, Streptococcus faecalis, and S.

aureus. Among these microorganisms, E. coli, S. aureus, S. sonnei, and S. Typhi

exhibited similar resistance to the UV-light and required approximately 7 mJ/cm2 energy

to get a 3 log10 reduction. However, the resistance exhibited by S. faecalis was higher

and required 1.4 times higher dose than the above mentioned microorganisms to get a 3

log10 reduction.

The effect of pulsed UV-light emission with low or high UV content on

inactivation of L. monocytogenes, E. coli, Salmonella Enteritidis, Psudeomonas

aeruginosa, Bacillus cereus, and S. aureus were investigated by Rowan et al. (1999). A

reduction of 2 or 6 log10 CFU/plate was obtained with 200 light pulses (duration of pulse

~100 ns) for low or high UV content, respectively. Treatment with three 360 µs duration

pulses resulted in more than 6 log10 CFU/ml reduction of B. subtilis for an energy input

of 1.2 J/cm2/s (Xenon, 2005).

Stermer and others (1987) investigated the effect of UV radiation on inactivation

of bacteria on lamb meat surfaces. With 4 mJ/cm2 energy, 99.9% of the naturally

occurring flora of lamb (mostly Pseudomonas, Micrococcus, and Staphylococcus species)

were inactivated. The bactericidal effectiveness of pulsed UV-light was investigated by

Bank and others (1990) on bacterial monolayers of S. epidermidis, Pseudomonas

aeruginosa, E. coli, S. aureus, or S. marcescens. A 60-s treatment time at 31 cm distance

from the light source (~4 J/m2) resulted in 6 to 7 log10 reduction of viable bacterial

population.

64

Aspergillus niger spores in corn meal were inactivated using a pulsed UV-system

(Jun et al., 2003). A 4.95 log10 reduction of A. niger on inoculated corn meal with a

treatment time of 100-s was achieved when the distance between the quartz window and

sample was kept at 8 cm with an input voltage of 3800 V. Sharma et al. (2003)

investigated the effect of pulsed UV-light on inactivation of E. coli O157:H7 on alfalfa

seeds. Reductions of 0.09 to 4.89 log10 CFU/g were obtained for various thickness and

treatment time combinations. Complete inactivation of E. coli O157:H7 was obtained

within 30-s treatment time, when the seed thickness was maintained at 1.02 mm, which

corresponds to 4.80 log10 CFU/g. Hillegas and Demirci (2003) studied the effect of

pulsed UV-light on inactivation of Clostridium sporogenes in honey. An 87.6%

reduction of Clostridium sporogenes was obtained when 2 mm depth of honey sample

was kept at 8 cm distance from the quartz window and treated with UV-light for 45-s,

whereas an 180-s treatment was required to achieve 89.4% reduction at 20 cm distance

from the quartz window. Krishnamurthy et al. (2005; chapter 3) reported that complete

inactivation of S. aureus (about 8 log10 CFU/ml reduction) can be obtained within 2 or 5 s

of pulsed UV-light treatment, when treated as agar seeded cells and cell suspension,

respectively. It was suggested that the pulsed UV-light treatment is non-thermal for a

short time applications (<5 s treatment). These studies demonstrate that pulsed UV-light

treatment can be utilized for the inactivation of S. aureus in both solid and liquid food

systems.

As the opacity of the liquid increases, penetration depth of UV-light in food

decreases. Therefore, only the top portion of the liquid and associated pathogen are well

exposed to UV-light because of poor penetration. So, pathogens may still survive in the

65

bottom portion and hence reduce the overall inactivation level of the pathogen in food.

Thus, better overall inactivation of a pathogen can be achieved in a less opaque food

matrix. For instance, Krishnamurthy et al. (2004; chapter 3) reported that a 5-s treatment

time was sufficient to achieve a 7 to 8 log10 reduction of S. aureus in 0.1 M phosphate

buffer (pH = 7.0). However, Hillegas and Demirci (2003) obtained less than one log10

reduction of C. sporogenes in honey treated for 45 s at 8-cm distance when a 2-mm depth

of sample was used. Jun et al. (2003) reported that voltage applied to the pulsed UV-light

system is an important parameter in pulsed UV-light treatment. Reductions of 1.35 and

4.95 log10 CFU/ml of A. niger spores were obtained in corn meal when treated for 100 s

and sample distance was kept as 8 cm for 2000 and 3800 V applied voltage, respectively.

Results of this study and other findings indicate that sample volume, treatment time,

sample distance from quartz window, opacity of the sample, and applied voltage are

some parameters which influence the inactivation of pathogens. By optimizing these

parameters one can effectively utilize pulsed UV-light treatment for a better inactivation

of pathogens.

As the literature review substantiates, there is not much research work done on the

inactivation of S. aureus in milk using pulsed UV-light treatment. Therefore, the overall

goal of this study was to investigate the possibility of using pulsed UV-light as an

alternative method for inactivation of S. aureus and determining the effect of various

parameters such as volume of sample, time of exposure, and distance from the UV-light

source on inactivation of S. aureus.

66


Preparation of inoculum


Microbiology Culture Collection. Stock cultures were maintained on tryptic soy agar

(TSA) (Difco) slants at 4oC. Also, stock cultures in 20% glycerol as a cryoprotectant

were maintained at -80oC in an ultra-low freezer (Sanyo Scientific Inc., Chatsworth, CA).

Cells were grown in 150 ml Tryptic Soy Broth (TSB; Difco, Sparks, MD) at 37oC for 24

h, and harvested by centrifugation (Sorvall STH750, Kendro Lab Products, Newtown,

CT) at 3,300 x g for 25 min at 4oC. The pellet was resuspended in 100 ml of raw milk

after decanting the supernatant to yield approximately 8 to 9 log10 CFU/mL of S. aureus.

Experimental design

A Box-Behnken surface response method design was utilized for experimental

design. This method has several advantages such as, reduced number of samples and

reduced number of replicates. This method recommends the treatments at the mid-points

of the edges (12 edges for 3 factors with 3 levels; hence, 12 data points) and the center of

the factor space (centre point is replicated three times; hence 12 + 3 = 15 data points

total) (Figure 4.1). A Box-Behnken design for three factor levels is 15 data points

whereas a full factorial design would have 27 data points (3 factors x 3 levels x 3

replicates = 27 treatments).

67

Figure 4.1. Surface response design

In general, the Box-Behnken design will have a combination of minimum,

maximum, and middle values specified for each factor. By using this method, one can

model the system effectively with fewer data points. MINITAB® (version 13.3; Minitab

Inc., State College, PA) statistical software was used to design the experiments.

Production of milk foam

Milk foam was produced by heating the milk to 65oC and adding 2% of whey

protein isolate (AlacenTM 895, NZMP, Lemoyne, PA). After mixing slowly the mixture

for 10 min, the milk was kept at 45oC for 10 min. Foam was created by bubbling air at 1

psi through the milk. The average temperature of the foam was 45 ± 2 oC. The foam was

then weighed and transported aseptically to pulsed UV-light treatment chamber in an

aluminum dish.

Sample volume

Distance

Treatment time

68


Pulsed UV-light treatment was carried out with SteriPulse®-XL 3000 Pulsed

Light Sterilization System (Xenon Corporation, Wilmington, MA; Figure 4.2). The

sterilization system generated 1.27 J/cm2 per pulse for an input voltage of 3,800 V and

with 3 pulses/s setting at 1.8 cm from the quartz window as per the manufacturer’s

specifications. It should be noted that the distance between the centre axis of the UV-

strobe and the quartz windows is 5.8 cm. The lamp produced a polychromatic radiation

in the wavelength range of 100 to 1100 nm, with 54% of the energy being in the UV-light

region (Panico, 2002). Volumes of 12, 30, and 48-mL of cell suspension in milk was

treated under pulsed UV-light for 30, 105, and 180 s according to the surface response

design (Table 4.1).

Figure 4.2. Schematic of Steripulse® XL-RS-3000 pulsed UV-light system.

The samples were placed inside the sterilization chamber in sterile aluminum

containers with vertical walls (7-cm diameter x 1.6-cm depth) at three sample distances

from quartz window levels: 8, 10.5, and 13-cm. The calculated depth levels

Control panel

Cooling

Milk sample

UV lamp

UV chamber

69

corresponding to 12, 30, and 48-mL of milk were 3.12, 7.79, and 12.47-mm, respectively.

The effect of pulsed UV-light on milk foam was also tested by evaluating the effect of the

following variables and levels, distance of milk foam from the quartz window (5, 8, and

11 cm), treatment time (30, 105, 180 s), and weight of milk foam (3, 5, and 7 g). Three

replications were performed for each condition.


Immediately after pulsed UV-light treatment, surviving populations of S. aureus

were enumerated. A 10 ml of 0.1% peptone was added gradually to milk foam in order

to recover the microorganism and serially diluted in 0.1% peptone water. In case of milk,

1-ml of sample was serially diluted using 0.1% peptone water. Both the samples were

spiral-plated on Baird-Parker agar by using Autoplate® 4000 (Spiral Biotech, Norwood,

MA). After incubating at 37oC for 24 h, the colonies were enumerated by Q-CountTM

(Spiral Biotech). The log10 reduction was calculated by determining the difference

between average log10 value of untreated samples and average log10 value of pulsed UV-

light treated samples. Also, enrichment was performed for zero or low counts of S.

aureus by transferring 1-ml of sample into 9-ml of TSB and incubating at 37oC for 24 h.

Temperature measurements

Milk temperature during pulsed UV-light treatment was monitored using K-type

thermocouples and the data were stored using a data-logger (Omegaette HH306, Omega

Engineering Inc., Stamford, CT).

70

Water bath studies

As the temperature of the milk increased during pulsed UV-light treatment, the

effect of heat treatment alone was investigated using a water bath study. A 50-ml milk

sample kept in a sterile 250-ml beaker (Kimax Model No. 14000, VWR international,

West Chester, PA) was inoculated with 1-ml of S. aureus culture and immediately placed

in the water batch (Aquabath Model No. 18800; Labline Instruments Inc., Melrose park,

IL) maintained at the room temperature. The inner diameter of the beaker was

approximately 6.5 cm, and the depth of milk was approximately 1.7 cm. The water in the

water bath was kept at least 1 cm above the milk surface to make sure that the milk was

always surrounded by hot water. The water bath was immediately turned on with the

temperature setting of 87oC, and the temperature was allowed to rise freely to reach the

target milk temperature of 85oC. Milk was constantly stirred in order to maintain

homogenous temperature. Three replications were used, and the temperature of the milk

was monitored regularly using a thermometer. A 1-ml milk sample was taken at regular

intervals (1, 2, 4, 8, 16, 32, and 64 min) and analyzed for surviving population of S.

aureus.

Energy measurements

The available broad band energy (100 to 1100 nm) at different tray levels of the

pulsed UV-light system was measured by using a radiometer (Ophir PE50, Ophir

Optronics Inc., Wilmington, MA). The pyroelectric detection head and the centre axis

were aligned and the detection head was placed at the centre. Measurements were taken

at this location, as all the experiments done throughout this study. The radiometer was

71

calibrated at 254 nm. The UV-light intensity at 254 nm was determined by comparing the

UV-light intensity obtained using another radiometer (SED240/ACT5/W detector head,

International lights, Newburyport, MA) as per the data given by Xenon Corporation

(Wilmington, MA).


Statistical significance was tested using the Surface Response Method using

MINITAB® software. A 95% confidence level was used to determine the significance.

Two independent data sets were used in model development and one independent data set

was used for validation for static milk treatment. For milk foam treatment, a surface

response model was developed with an independent data set.


The effects of sample volume, treatment time, and distance from the light source

were investigated for milk foam. The effects of foam weight, treatment time, and

distance were investigated for milk foam. A surface response methodology design was

used to design the experiments.

Static milk treatment

The log10 reductions obtained varied from 0.16 to 8.55 log10 CFU/ml (Table 4.1).

This demonstrated the ability of pulsed UV-light to inactivate S. aureus. The effects of

treatment time and time*volume interaction were significant (p<0.05). The log10

reduction increased as the treatment time increased, sample volume decreased, or the

72

sample distance from the quartz window decreased. Maximum log reductions of 8.55

log10 CFU/ml were obtained for 1) 8-cm sample distance from quartz window, 30-ml

sample volume, and 180-s treatment time combination and 2) 10.5-cm sample distance

from quartz window, 12-ml sample volume, and 180-s treatment time combination.

Table 4.1. Surface response method analysis of pulsed UV-light treatment of milk.

Distance (cm)

Time (s)

Volume (ml)

Log10 reduction1

Validation log10 reduction

(experimental) 8 30 30 1.13 ± 0.45 0.89 8 105 12 2.13 ± 0.28 1.87 8 105 48 0.16 ± 0.07 0.12 8 180 30 8.55 ± 0.27* 8.63

10.5 30 12 0.19 ± 0.01 0.23 10.5 30 48 0.93 ± 0.71 1.01 10.5 105 30 1.68 ± 0.37 1.50 10.5 105 30 0.62 ± 0.31 0.78 10.5 105 30 1.04 ± 0.34 1.35 10.5 180 12 8.55 ± 0.27* 8.63 10.5 180 48 0.94 ± 0.42 0.79 13 30 30 0.14 ± 0.06 0.21 13 105 12 3.01 ± 1.00 2.89 13 105 48 0.19 ± 0.14 0.31 13 180 30 0.61 ± 0.58 0.46

* Complete inactivation of Staphylococcus aureus was obtained at these conditions (Enrichments were negative) 1Mean and standard deviation of two replications are given. Mean was used to develop the model.

Effect of sample volume

As the volume of the sample decreased the log10 reduction increased at longer

treatment times (Figure 4.3A). For example, pulsed UV-light treatment of S. aureus at

10.5- cm distance and 180-s treatment time resulted in 8.55 and 0.94 log10 CFU/ml

reductions for 12 and 48-ml sample volumes, respectively. The treatment time*volume of

73

sample interaction was significant (p<0.05). In other words, the effect of treatment

volume also depends upon the treatment time. For example, log10 reductions of 1.13 and

8.55 log10 CFU/ml were obtained for 30 and 180-s treatments, respectively, when sample

distance was kept at 8 cm and the sample volume was 30 ml. Also at lower volumes,

there is a rapid increase in log10 reduction for increase in treatment time, when compared

to higher volumes because of poor penetration capacity of UV-light.

Effect of treatment time

As the treatment time increased, the log10 reduction increased (p<0.05) because of

increased cumulative energy absorption by the bacteria (Figure 4.3B). Also, the

distance*time interaction was significant (p<0.05). Reductions of 0.19 and 8.55 log10

CFU/ml were obtained when treated for 30 s and 180 s, respectively, at 10.5-cm sample

distance from quartz window and 12-ml sample volume. Data clearly show that there is

an exponential relationship between log10 reduction and treatment time. This can be

verified by the rapid increase in log10 reduction with the time of exposure.

Effect of distance

The amount of energy received by the sample decreased as the sample distance

from the quartz window increased (Figure 4.3B). When compared to other distances

from the UV-strobe, the 8-cm distance exhibited higher log10 reduction, since the sample

was closer to the UV lamp than samples at 10.5 and 13-cm; the samples hence received

more energy.

74

5040

30-140

0

Volume (ml)

123456

90

78

20140

log reduction

10190Time (sec)

5040

308

-1 Volume (ml)

0

9

1

2

3

10

4

2011

log reduction

12 1310Distance (cm)

A

B

190140

8-10

Time (sec)

1

909

2345

10

67

11

log reduction

12 4013Distance (cm)

C

Figure 4.3. Inactivation of S. aureus in milk: A) Interaction of treatment time and sample volume (hold value: distance: 10.5 cm), B) Interaction of distance from quartz window

and treatment time (hold value: volume: 30 ml), and C) Interaction of distance from quartz window and sample volume (hold value: time: 105 s).

75

For instance, 180-s treatment of 30-ml sample resulted in 8.55 and 0.61 log10 CFU/ml

reduction when treated at 8 and 13-cm distance from the quartz window, respectively.

The effect of distance was found to have a significant impact on log10 reduction

during longer treatment time compared to shorter treatment times (

Figure 4.3B). The log10 reduction increased as the volume of sample and the

sample distance from quartz window decreased (Figure 4.3C).

These results are compared with studies of Smith et al. (2003) and Bank et al.

(2000). Smith et al. (2003) obtained ~2.0 log10 reduction of S. marcescens when

inoculated in raw bulk tank milk after 28-s treatment time (6.6 J/cm2 dose level) with a

pulsed UV laser light. Bank et al. (2000) reported that a 60-s treatment time at 31-cm

distance from the light source (approximately 4 x10-4 J/cm2) resulted in 6 to 7 log10

reduction of S. aureus on seeded TSA plates.

Temperature profile during pulsed UV-light treatment

The pulsed UV-light treatment for inactivation of microorganisms is considered a

non-thermal process for short times (less than 5 or 10-s) (chapter 3). However, increase

in cumulative energy results in temperature increase. Therefore, as treatment time

increased, the temperature increased gradually (Figure 4.4). The temperature of the

sample was 28oC, 58.1oC, and 91.2oC when treated for 10, 60, and 180 s, respectively,

when a sample volume of 12-mL was kept at 8 cm distance. Also, as the distance from

the quartz window decreases, the temperature increases as the energy absorption

increases because of more energy available to the sample. Sample temperatures were

72.3oC, 69.6oC, and 38.6oC after 100 s treatment when the sample volume was 30-mL for

76

sample distances of 8, 10.5, and 13-cm, respectively. Similarly, an increase in sample

volume resulted in a decrease in temperature rise. For example, a 180-s treatment at 8-

cm sample distance from the quartz window resulted in 91.2oC, 73.2oC, and 57oC sample

temperatures, when the sample volumes were 12, 30, and 48 ml, respectively.

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120 140 160 180Time (sec)

Tem

pera

ture

(o C)

8 cm 12 ml

10.5 cm 12 ml

8 cm 30 ml

10.5 cm 30 ml

13 cm 48 ml

8 cm 48 ml13 cm 30 ml

10.5 cm 48 ml

13 cm 12 ml

Figure 4.4. Temperature profile during pulsed UV-light treatment

Heat treatment of S. aureus in water bath

There was a considerable increase in the temperature of a milk sample during

pulsed UV-light treatment. Therefore, the inactivation also might have been partly

contributed to the temperature increase. In order to investigate this possibility, milk

inoculated with S. aureus was heated in a water bath to the same temperature achieved

during pulsed UV-light treatment, which was increased to about 85oC after a 3 min of

pulsed UV-light treatment.

The temperature increase during the pulsed UV-light treatment was gradual.

Therefore, the amount of time required for the gradual increase in temperature for

77

inactivation of S. aureus was investigated. The water bath temperature was set at 88oC,

so that the temperature of the milk sample can reach up to 85oC gradually. It took 64 min

for complete inactivation of S. aureus (Table 4.2). The temperature reached 85oC after

40 min.

Table 4.2. Inactivation of S. aureus by constant increase in temperature.

Log10 reduction Growth after enrichment Time (min)

2 0.75 Yes 4 0.70 Yes 8 0.62 Yes 16 1.20 Yes 32 3.21 Yes 64 8.12 No

These results indicate that the temperature rise in milk also contributed to

inactivation of S. aureus. However, it took about 3 min for pulsed UV-light treatment,

whereas it took more than 40 min for the heat treatment in a water bath.

Surface response model and validation for milk

A full quadratic surface response model with constant, linear, interactions, and

squared terms was developed. Variables used in the model were sample distance from

the quartz window (Distance “D”, cm), treatment time (Time “T”, s), and volume of

sample (Volume “V”, ml). Response surface modeling was done using MINITAB with

uncoded units and the following regression equation was obtained.

Log10 reduction = β0 + β1D+ β2T+ β3V+ β22T2 + β12DT+ β13DV+ β23TV+ ε.

Where, ε = Error and β0, β1, β2, β3, β22, β12, β13, and β23 are coefficients (Table 4.3)

78

D2 and V2 terms are not included in the model, because they were not significant.

Although T2 term was not significant, it was used in the model because the Time (T) term

was significant.

Table 4.3. Regression coefficients of response surface model for inactivation of S. aurues in milk.

Term Coefficient p value β0 -8.83684 0.247 β1 0.71367 0.296 β2 0.11970 0.047* β3 0.13097 0.471 β22 0.00024 0.097 β12 -0.00927 0.040 β13 -0.00472 0.767 β23 -0.00155 0.019

*Bold values indicate terms which are statistically significant (p<0.05).

Defining optimum or maximum log reduction: The model predicted a

mathematically optimum condition of 8 cm distance, 12 ml volume, and 180 s treatment

time, yielding an estimated reduction of 10.74 log10 CFU/ml. Not surprisingly, this is the

closest distance, smallest volume, and longest time the model was allowed to consider

and was a more severe treatment than any condition in the experiment. Complete

inactivation of S. aureus was obtained when the UV-light treatment was conducted at the

above mentioned optimum condition, which corresponds to 8.70 log10 CFU/ml reduction.

The observed log10 reduction (8.70 log10 CFU/ml) was less than the predicted log10

reduction (10.74 log10 CFU/ml) because of the limitation of maximum bacterial density.

As mentioned earlier, as the volume of the sample decreases the log10 reduction

increases. These indicate that one should expect to get complete inactivation of S. aureus

at the model generated optimal condition.

79

An R2 value (coefficient of determination) of 0.88 was obtained with this model,

which implies that 88% of the sample variation was taken into account by the model.

Only treatment time, time*distance interaction, and time*volume interaction were

significant (p<0.05) (Table 4.3). An independent set of data was collected in order to

validate the model developed. The predicted data reasonably agrees with the

experimental data with an R2 of 0.87 (Figure 4.5) and a slope of 0.90, when the trend line

passes through the origin.

y = 0.9xR2 = 0.8664

-2.00

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 1 2 3 4 5 6 7 8 9

Experimental reduction (log10 CFU/ml)

Pred

icte

d re

duct

ion

(log 1

0 CFU

/ml)

Trendline passing through origin for given data set

Figure 4.5. Validation of the response surface model

A perfect fit would have an R2 of 1.0 and a slope of 1.0 for the same condition.

When the data points were forced to fit the perfect fit (experimental = predicted) line, the

model yielded an R2 of 0.85, which indicates that predictive ability of the model is

reasonably good.

80

4.5. Inactivation of S. aureus in milk foam

UV-light has a poor penetration capacity. Therefore, it is necessary to facilitate

vigorous mixing of a milk sample in order to expose most of the sample to UV-light

during continuous flow treatment of milk. Foam is produced during this vigorous mixing

and it may behave differently as the composition is different from that of the regular milk

sample. Therefore, Inactivation of S. aureus in milk foam was studied in order to

investigate the effect of UV-light on foam. Reductions of 1.05 to 6.61 log10 CFU/g were

obtained (Table 4.4 and Figure 4.6).

Table 4.4. Inactivation of S. aureus in milk foam.

Distance (cm)

Time (s)

Weight (g)

Reduction (Log10

CFU/g)1

Validation reduction (Log10 CFU/g -experimental)

5 30 5 1.19±0.01 1.17 5 105 3 6.61±0.11* 7.10* 5 105 7 5.00±0.26 4.94 5 180 5 6.39±0.11* 6.88* 8 30 3 1.67±0.05 1.68 8 30 7 1.05±0.23 1.31 8 105 5 6.39±0.11* 6.88* 8 105 5 6.39±0.11* 6.88* 8 105 5 6.39±0.11* 6.88* 8 180 3 6.61±0.11* 7.10* 8 180 7 6.24±0.11* 6.73* 11 30 5 1.70±0.33 1.72 11 105 3 6.61±0.11* 7.10* 11 105 7 2.13±0.44 2.53 11 180 5 6.39±0.11* 6.88*

*No growth observed on agar plates after incubation. Further enrichment was positive for most of the samples indicating sublethal injury and cells were able to repair themselves.

81

200150

Log reduction

2

4

100

6

T ime (sec)

8

5.0 507.5 10.0Distance (cm)

A

7.56.0

Log reduction

3

4

5

6

Weight (g)4.55.07.5 3.010.0

Distance (cm)

B

200150

Log reduction

0

2

4

100

6

Time (sec)3.0 504.5 6.0 7.5Weight (g)

C

Figure 4.6. Inactivation of S. aureus in milk foam: A) Interaction of distance and

treatment time (hold value: weight of foam: 5 g), B) Interaction of distance from quartz window and weight of foam (hold value: treatment time: 105 s), and C) Interaction of

foam weight and treatment time (hold value: distance: 8 cm).

82

As expected, increase in treatment time resulted in higher inactivation of S.

aureus (Figure 4.6A). The effect of treatment time on inactivation of S. aureus was

statistically significant (p<0.05). For instance, reductions of 1.19 and 6.39 log10 CFU/g

were obtained for 30 and 180 s treatment, respectively, when the distance from the quartz

window was 5 cm for 5 g of milk foam. The cumulative energy absorbed by S. aureus is

increased as the treatment time increases, resulting in a higher probability of photon

absorption for effective inactivation.

Weight of the milk foam did not have a significant effect on inactivation of S.

aureus (p > 0.05). For example, reductions of 6.61 and 6.24 log10 CFU/g were obtained

after treatment of 3 and 7 g of milk foam, respectively, when the samples were treated for

180 s at 8 cm distance from the quartz window (Figure 4.6C). As the UV-light can easily

penetrate through the foam because of its structure, increasing the volume of foam did

not change the inactivation significantly.

In general, increasing the distance of the sample from the quartz window did not

significantly affect the inactivation as the effect of treatment time was predominant

(Figure 4.6A) (p>0.05).

Surface response model and validation for milk foam

A full quadratic equation was developed for the response surface model using

MINITAB with uncoded units. The following variables are used in developing the

model, 1) sample distance from the quartz window (Distance “D”, cm), 2) treatment time

(Time “T”, s), and 3) weight of sample (Weight “W”, g). The response surface model

obtained was as follows:

83

Log10 reduction = β0 + β1D+ β2T+ β3W+ β22T2 + β12DT+ β13DW+ β23TV+ ε.

where, ε = Error and β0, β1, β2, β3, β22, β12, β13, and β23 are coefficients (Table 4.5).

Table 4.5. Regression coefficients of response surface model for inactivation of S. aurues

in milk foam.

Term Coefficient P value Constant β0 -11.4171 0.132 Distance β1 1.6927 0.156

Treatment time β2 0.1043 0.019* Weight β3 2.1202 0.207

Distance*Distance β11 -0.0709 0.261 Treatment time*Treatment

time β22 -0.0003 0.015

Weight*weight β33 -0.1651 0.247 Distance* treatment time

β12 -0.0006 0.803

Distance*weight β13 -0.1194 0.199 Treatment time*weight β23 0.0004 0.902

*Bold values indicate terms which are statistically significant (p<0.05)

The surface response model had an R2 of 94% indicating that the model was able

to explain 94% of the variation in the model. The developed model predicted the

inactivation rate within a reasonable tolerance as indicated by an R2 of ~0.93 and slope of

~0.99 (Figure 4.7).

Energy measurement

It is very important to know the actual energy available at the surface of food

material since a considerable portion of the energy is wasted during pulsed UV-light

treatment. As different pulsed UV-light systems have different configurations, the

energy available is not the same for the same amount of exposure at the same distance.

Therefore, by specifying the energy available for microbial inactivation, one can

84

standardize the pulsed UV-light inactivation. The broadband energy measurements for

various distances from the quartz window are given in Table 4.6. As the distance from

the quartz window increases, the energy available is decreased gradually. For instance,

energy levels of 0.40, 0.33, and 0.19 J/cm2 were available at 3, 8, and 13 cm distances

from the quartz window, respectively.

y = 0.9897xR2 = 0.9295

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Log10 reduction (experimental)

Log 1

0 red

uctio

n (m

odel

pre

dict

ed)

Figure 4.7. Experimental vs. predicted log10 reduction of S. aureus in milk foam.

Absorption at 254 nm may be an indicator of DNA absorption. Therefore, it is

important to know what portion of the energy is available at 254 nm, which contributes to

the photochemical changes in the DNA. Only 2% of the total broadband energy is

available in the 254 nm wavelength region in the pulsed UV-light system. For instance,

the broadband energy (100 to 1100 nm range) at 3 cm distance from the quartz window

was 0.40 J/cm2, whereas only 0.008 J/cm2 energy was present 254 nm.

85

Table 4.6. Broadband energy measurement during pulsed UV-light treatment1.

Approximate distance from

Quartz window2 (cm)

Energy3 (J/pulse)

Energy4 (J/cm2 per

pulse)

Energy at 254 nm (J/cm2)

3.0 7.19 0.40 0.008 5.0 6.48 0.36 0.007 7.0 5.93 0.33 0.007 8.0 5.93 0.33 0.007 10.5 4.85 0.27 0.005 13.0 3.47 0.19 0.004

1Radiometer was calibrated at 254 nm and measured the broadband energy in the wavelength range of 100 to 1100 nm. 2The distance between the quartz window and the centre axis of the UV-strobe is 5.8 cm. 3Energy was averaged over 30 pulses; three independent measurements were taken and average is reported. 4Surface area of the radiometer detector head was 18.096 cm2.

4.6. Conclusions

The potential of pulsed UV-light as an alternative process for the inactivation of

S. aureus in milk was demonstrated. A surface response model was developed and

validated successfully. Pulsed UV-light is highly effective in reducing the pathogens in

milk, which can be demonstrated by the complete inactivation of S. aureus obtained after

180-s of treatment time in two cases (Table 4.1) and as predicted by model. Though the

holding time for HTST pasteurization is at 71oC for 15 s, the total time required for the

pasteurization process will be several minutes as the raw milk at 4oC has to be preheated

in the regenerator section and heated by steam or hot water to achieve the required

temperature. However, pulsed UV-light treatment takes about 3 minute for inactivation

of pathogens even though milk was under static conditions. Pulsed UV-light was also

effective on inactivation of S. aureus on milk foam. The temperature increase during

pulsed UV-light treatment also played a significant role on inactivation of S. aureus.

86

Therefore, both temperature increase and photochemical changes account for microbial

inactivation during longer treatment. However, for a short period of pulsed UV-light

exposure, the effect of temperature increase is negligible and hence the inactivation

occurs mainly because of photochemical changes in DNA. If the system is designed for

continuous milk pasteurization then this can be validated. Further research needs to be

done to find an optimum condition for the inactivation of S. aureus for continuous flow

conditions to represent commercial cases. Effect of the pulsed UV-light process on the

quality and nutritional attributes of milk must be examined to assess any adverse effects

of UV-light.

4.7. References

Bank, H.L., J.L. Schmehl, and R.J. Dratch. 1990. Bacteriocidal effectiveness of


Bramley, A.J. and C.H. McKinnon. 1990. The microbiology of raw milk. In Dairy

Microbiology: The Microbiology of Milk. Vol. 1, R.K. Robinson, ed. New York,

NY: Elsevier Applied Science.

Buzby, J.C., T. Roberts, C.T.J. Lin, and J.M. MacDonald. 1996. Bacterial foodborne

disease: Medical costs and productivity loses. AER report No. 741. Washington,

DC: Economic research services – United States Department of Agriculture.



Applied Nutrition – Food and Drug Administration. Available at:

http://vm.cfsan.fda.gov/~comm/ ift-puls.html. Accessed 19 April 2006.



Appl. Environ. Microbiol. 49(6):1361-1365.

87

http://vm.cfsan.fda.gov/%7Ecomm/%20ift-puls.html

Crutchfield, S.R. and T. Roberts. 2000. Food safety efforts accelerate in the 1990’s. Food

Review. 23(3):44-49.

Dunn, J.., T. Ott, and W. Clark. 1995. Pulsed light treatment of food and packaging.

Food Technol. 49:95-98.

Hillegas, S. L. and A. Demirci. 2003. Inactivation of Clostridium sporogenes in clover

honey by pulsed UV-light treatment. Manuscript FP 03-009. CIGR J. AE Sci. Res.

Dev. Vol. V. 7 pp.


55(12):16.

IDFA. 2001. Milk Facts. Washington, D.C.: International Dairy Foods Association.

Jun, S., J. Irudayaraj, A. Demirci, and D. Geiser. 2003. Pulsed UV-light treatment of corn

meal for inactivation of Aspergillus niger spores. Int. J. Food Sci. Technol.

38:883-888.

Kime, Z.R. 1980. Sunlight. Penryn, CA: World Health Publications.







1587.

Mead, P.S., L. Slutsker, V. Dietz, L.F. McCaig, J.S. Bresee, C. Shapiro, P.M. Griffin, and

R.V. Tauxe. 1999. Food-related illnesses and death in the United States. Emerg.

Infect. Dis. 5(5):607-625.

Miller, R., W. Jeffrey, D. Mitchell, and M. Elasri. 1999. Bacterial responses to ultraviolet

light. ASM news. 65(8):534-541.




88

Panico, L.R. 2002. Instantaneous surface sanitation with pulsed UV. Brussels, Belgium:

Hygienic coatings global conference. Available at: http://www.xenon-corp.com/

press/images/SurfaceSanitization.pdf.



modeling. J. Food Sci. 68:1448-1453.

Smith, W.L., M.C. Lagunas-Solar, and J. S. Cullor. 2002. Use of pulsed ultraviolet light

for the cold pasteurization of bovine milk. J. Food Prot. 65:1480-1482.


effective bactericide for fresh meat. J. Food Prot. 50:108-111.

Xenon, 2005. Pulsed UV treatment for sterilization and decontamination. Woburn: MA:

Xenon Corporation.

89

http://www.xenon-corp.com/%20press/images/SurfaceSanitization.pdf

http://www.xenon-corp.com/%20press/images/SurfaceSanitization.pdf

5. Inactivation of Staphylococcus aureus in Milk Using

Flow-through Pulsed UV-Light Treatment System

5.1. Abstract

This study investigated the efficacy of pulsed UV-light for continuous-flow milk

treatment for the inactivation of Staphylococcus aureus, pathogenic microorganism

frequently associated with milk. Pulsed UV-light is a novel technology which can be

used for inactivation of this pathogen in milk in a short time. Pulsed UV-light damages

the DNA of the bacteria by forming thymine dimers which leads to bacterial death. The

effect of sample distance from the UV-light source, number of passes, and flow rate were

investigated. A response surface method was used for design and analysis of the

experiments. Milk was treated at 5, 8, or 11 cm distance from UV-light strobe at 20, 30,

or 40 ml/min flow rate and treated up to three times by recirculation of milk to find the

effect of number of passes on inactivation efficiency. Log10 reductions varied from 0.55

to 7.26 log10 CFU/ml. Complete inactivation was obtained in two cases and growth was

not observed mostly following the enrichment protocol. A response surface method was

used for the design of the experiments. The predicted data were in agreement with the

experimental data. Overall, this work demonstrated that pulsed UV-light has a potential

for inactivation of milk pathogens.

90

5.2. Introduction


325,000 hospitalizations, and 5,200 deaths annually in the United States (Mead et al.,

1999). There have been several outbreaks associated with milk or milk products. Milk

may contain spoilage microorganisms and pathogenic microorganisms because of

improper handling and contamination from both inside and outside the udder (Bramley

and McKinnon, 1990). Therefore, it is necessary to process milk to inactivate these

microorganisms. Conventionally, milk pasteurization is done using heat exchangers.

However, pasteurization is a high energy demanding process with high capital and

operational costs involved. Therefore, there is a need for an alternative processing

method that is simple, cost-effective, and has high inactivation efficiency.

The Center for Disease Control and Prevention (CDC) estimates that there have

been 17,248 and 1,413 cases of Staphylococcus aureus reported during 1973-87 and

1983-87, respectively, which accounted for 14% and 1.6% of all the cases because of

bacterial pathogens (Bean and Griffin, 1990; Bean et al., 1990; Olsen et al., 2000).

Pulsed UV-light treatment could serve as a potential method for the inactivation of S.

aureus in milk. UV-light has been used as a bactericide as early as 1928 (Xenon, 2003).

UV-light is the portion of the electromagnetic spectrum ranging from 100 to 400 nm

wavelengths and possesses germicidal properties in the wavelength region of 220 to 280

nm. UV-light damages the DNA by forming thymine dimers, which leads to cell death

(Bank et al., 1990; Miller et al., 1999).

There are two modes of application of UV-light: continuous or pulsed UV-light

mode. Continuous UV-light is the conventional one, which delivers the UV-light in a

91

continuous mode. In the pulsed UV-light mode, the UV-light is stored in a capacitor and

released as intermittent pulses, thus increasing the instantaneous energy. Therefore,

pulsed UV-light treatment is a more effective and rapid way of inactivating

microorganisms than continuous UV-light sources since the energy is multiplied many

fold (Dunn, 1995; CFSAN-FDA, 2000). McDonald et al. (2000) reported that the almost

identical level of inactivation of Bacillus subtilis was obtained with 40 J/m2 of pulsed

UV-light source and 80 J/m2 continuous UV-light sources.

Bank et al. (1990) reported that a 60-s pulsed UV-light treatment at 31 cm

distance from the light source (~4 J/m2) resulted in 6 to 7 log10 reduction of viable

bacterial populations of Staphylococcus epideridis, Pseudomonas aeruginosa,

Escherichia coli, S. aureus, or Serratia marcescens on Trypticase soy agar plates.

Jun et al. (2003) investigated the effect of pulsed UV-light on inactivation of

Aspergillus niger spores in corn meal and reported a 4.95 log10 reduction for a treatment

time of 100 s when the distance between the quartz window and sample was kept at 8 cm

and the input voltage was 3800 V. Sharma et al. (2003) investigated the effect of pulsed

UV-light on inactivation of E. coli O157:H7 on alfalfa seeds. Reductions of 0.09 to 4.89

log10 CFU/g were obtained for various thickness and treatment time combinations. Four

thicknesses (1.02, 1.92, 3.61, and 6.25 mm) and 7 treatment times (5, 10, 30, 45, 60, 75,

and 90 s) were used in the experiment. Complete inactivation of E. coli O157:H7 was

obtained within 30 s of the treatment time, when the seed thickness was maintained at

1.02 mm, which corresponds to 4.80 log10 CFU/g. Increase in treatment time resulted in

higher log10 reduction for all the thicknesses (p<0.05). Hillegas and Demirci (2003)

obtained 87.6% reduction of Clostridium sporogenes in honey when 2 mm depth of

92

honey sample was kept at 8 cm distance from the quartz window and treated with pulsed

UV-light for 45 s.

Krishnamurthy et al. (2004; chapter 3) reported that pulsed UV-light is a non-

thermal process during a short period treatment (<10 s) and obtained more than 8 log10

reduction of S. aureus in a phosphate buffer or agar seed cells within 5 s treatment time.

They also reported that complete inactivation of S. aureus, which corresponds to 8.55

log10 CFU/ml, was obtained in two cases, when static milk samples were treated with

pulsed UV-light (Chapter 4). The conditions at which complete inactivation was

obtained were as follows: 1) 8 cm sample distance from the quartz window, 30 ml sample

volume, and 180 s treatment time combination, and 2) 10.5 cm sample distance from the

quartz window, 12 ml sample volume, and 180 s treatment time combination.

The significance of the safety of food products reveals ample potential for the

proposed study on continuous milk treatment using pulsed UV-light, which could be

implemented in a food plant for continuous processing. Understanding the efficacy of

pulsed UV-light in pathogen inactivation and developing test protocols in a beta-site will

help in the development of a commercial UV system for use in a food plant. In this

study, the inactivation of artificially inoculated S. aureus in milk with respect to key

design parameters was demonstrated.

93




Microbiology Culture Collection and maintained at 4oC on tryptic soy agar (TSA; Difco,

Sparks, MD) slants. Cells were grown in 150 ml Tryptic Soy Broth (TSB; Difco) at 37oC

for 24 h, and harvested by centrifugation (Sorvall STH750, Kendro Lab Products,

Newtown, CT) at 3,300 x g for 25 min at 4oC and the supernatant was decanted.

Raw milk was obtained from the University Creamery or dairy farm at

Pennsylvania State University and stored in the refrigerator at 4oC for a maximum of

three days. The centrifuged S. aureus cell pellet from the previous step was suspended in

100 ml raw milk to yield approximately 8 to 9 log10 CFU/ml and was vortexed vigorously

to ensure homogeneity.

Experimental design

In order to reduce the number of treatments, a response surface method (Box-

Behnken) design was used (MINITAB® version 13.3; Minitab Inc., State College, PA).

Distance of the sample from the UV-light strobe (5-11 cm), number of passes (1-3

passes), and flow rate (20-40 ml/min) are the three variables used in the design.


Pulsed UV-light treatment was carried out with the SteriPulse®-XL 3000 pulsed

light sterilization system (Figure 5.1; Xenon corporation, Woburn, MA), which produced

polychromatic radiation in the wavelength range of 100 to 1100 nm, with 54% of the

94

energy being in the UV-light region. The sterilization system generated three pulses per

second and 1.27 J/cm2 per pulse at 1.8 cm from the quartz window surface for an input

voltage of 3,800 V as per the manufacturer. A flow-through system was installed in the

pulsed UV-light chamber (Figure 5.2).

Figure 5.1. SteriPulse®-XL 3000 Pulsed UV-light sterilization system (Xenon Corporation®)

UV-light treated milk

Pump with variable flow rate

Milk inoculated with S. aureus

V- Groove reflector

UV-light chamberUV-light lamp

UV-light treated milk



V- Groove reflector




V- Groove reflector


Figure 5.2. Schematic diagram of continuous milk treatment system.

95

Milk inoculated with S. aureus was pumped through a quartz tube (1.14-cm i.d.,

1.475-cm o.d.) exposed to pulsed UV-light using a peristaltic pump (Model No. 7524-50;

Masterflex® L/S®, Barnant Co., Barrington, IL). Only 28 cm of the quartz tube length

was exposed to UV-light, and the rest of the tubing including flexible connection tubing

was covered with aluminum foil to block UV-light exposure. Also, a V-groove reflector

with polished surface was used to hold the quartz tube in order to reflect the UV-light

back to the quartz tube and hence increase the energy absorbed by the sample. The V

groove reflector had angles of approximately 56 and 117o (Figure 5.4). The reflector

consisted of a sharp channel to hold the quartz tube in place and the top portion of the

reflector was more flat to avoid any shadow effect. The reflector was designed by the

manufacturer of the pulsed UV-light system (Xenon corporation, Wilmington, MA)

based on trial and error to maximize the UV-light energy absorption.

Figure 5.3. V-groove reflector

117o

56o

Both the angles play an important role in determining how much energy can be

reflected back to the sample. Typically an elipse shaped reflector would serve the

purpose better as all the energy can be focused back to the sample. Further investigation

on the design of the V-groove reflector is warranted for optimization of the pulsed UV-

light processing.

96

The central axis of the quartz tube was aligned with that of the UV-light lamp in

order to maximize the energy absorption. The receiving container was held at an altitude

of 47 cm in order to reduce the bubble formation during pumping. Depending upon the

surface response design, milk was treated up to three times by recirculation.

Experimental design

A surface response design was used in this study (Table 5.1) with the following

variables: 1) distance of sample from the quartz window, 2) number of passes, and 3)

flow rate. A Box-Behnken design was used with MNITAB® (version 13.3; Minitab Inc.,

State College, PA) statistical software.

Table 5.1. Box-Behnken response surface method design parameters.

Variable Minimum Maximum

Distance from Quartz window*

5 cm 11 cm

Number of passes 1 3 Flow rate 20 ml/min 40 ml/min

* Distance between quartz window and the center axis of the UV-lamp is 5.8 cm.

Temperature measurement

The temperature profile of the milk was monitored using a K-type thermocouple

connected with the data logger (Omegaette HH306, Omega Engineering Inc., Stamford,

CT). The thermocouples were inserted in the tubing immediately outside the pulsed UV-

light system at the same distance to measure the inlet and outlet temperatures. The bulk

temperature of the milk was also measured using a thermometer after pulsed UV-light

treatment by adequately mixing to ensure homogenous temperature.

97


Populations of S. aureus were enumerated before and immediately after pulsed

UV-light treatment. A 1-ml sample was serially diluted using 0.1% peptone water and

spiral-plated on Baird-Parker agar using Autoplate® 4000 (Spiral Biotech, Norwood,

MA). The colonies were enumerated using Q-CountTM (Spiral Biotech) following

incubation at 37oC for 24 h. The log10 reduction was calculated by determining the

difference between average log10 value of untreated samples and average log10 value of

pulsed UV-light treated samples. Enrichment protocol was followed for zero or low

counts of S. aureus by transferring 1-ml of pulsed UV-light treated milk sample into 9-ml

of TSB and incubating at 37oC for 24-48 h.


MINITAB® software was used to test the statistical significance of the

parameters. A 95% confidence level was used to determine the significance. Three

replications were done and two independent data sets were used in the model

development and one independent data set was used for the validation of the developed

model.


For continuous treatment of milk by pulsed UV, the variables tested were: 1)

distance of sample from the quartz window, 2) number of passes, and 3) flow rate. The

log10 reduction obtained from the pulsed UV-light treatment ranged from 0.55 to 7.26

log10 CFU/ml (Table 5.2). Complete inactivation of S. aureus was obtained at 1) 8 cm

98

sample distance from quartz window, single pass, and 20 ml/min flow rate, and 2) 11 cm

sample distance from quartz window, 2 passes, and 20 ml/min flow rate combinations.

Table 5.2. Log10 reductions of S. aureus in milk by pulsed UV-light treatment.

Run Order Distance Passes Flow rate Log10 reduction++

Validation data set

(experimental) 1 11 3 30 0.63 ± 0.40 0.48 2 8 3 20 2.07 ± 0.33 1.56 3 8 1 40 0.55 ± 0.27 0.65 4 8 1 20 7.23 ± 0.36* 7.09* 5 11 2 40 0.73 ± 0.26 0.43 6 5 2 40 0.77 ± 0.06 0.76 7 5 2 20 2.48 ± 0.66 2.11 8 8 2 30 4.72 ± 0.69 4.48 9 8 2 30 5.87 ± 0.08 5.60 10 5 3 30 2.40 ± 0.01 2.63 11 11 1 30 0.72 ± 0.18 0.99 12 5 1 30 1.25 ± 0.07 0.98 13 11 2 20 7.26 ± 0.32* 7.09* 14 8 3 40 1.36 ± 0.10 1.17 15 8 2 30 4.76 ± 0.35 4.62

*Indicates complete inactivation. In most of the cases, there was no growth even after enrichment. ++Three replications were performed. Two used for model development and one for model validation. Mean and standard deviation are given. Mean was used for the model development. Statistical analysis revealed that the sample distance from the UV-light source is

the only variable which has statistical significance at the 95% confidence level (p<0.05).

However, the distance*distance interaction and passes*passes interaction had a slightly

higher p values than the threshold (p=0.05), where the corresponding p values are 0.053

and 0.052, respectively.

The log10 reduction varied as a function of the distance from the quartz window,

wherein the microbial inactivation curve resembles a bell shaped curve (Figure 5.4).

99

3

log reduction

1.02

2.5

4.0

Passes

5.5

5.07.5 110.0

DistanceHold Values

Flow rate 30

Figure 5.4. Surface plot of log10 reduction vs. passes and distance.

Though, the log10 reduction increased as the distance increased, after reaching

certain distance, log10 reduction decreased, indicating that maximum reduction can be

obtained within the limits of the variable. For instance the log10 reductions of 1.25 and

0.72 log10 CFU/ml were obtained at 5 and 11 cm distance from UV-light source,

respectively, when the flow rate was 30 ml/min in a single pass UV-light treatment

(Table 5.2).

The log10 reduction at 8 cm distance from the quartz window, 30 ml/min flow rate

and single pass was 3.66 log10 CFU/ml. This clearly shows that the maximum log10

reduction was obtained at the middle point of the surface plot where the number of passes

was 2, distance from UV-light source was 8 cm and the flow rate was 30 ml/min.

As expected, the lower flow rate resulted in better inactivation in most of the

cases as the absorbed energy is high because of longer residence time (Figures 5.5 and

5.6). The residence time corresponding to the flow rates of 20, 30, and 40 ml/min are

5.52, 3.68, and 2.76 minutes, respectively resulting in a processing volume of ~110 ml.

100

The volume of milk processed for a given treatment time increased in continuous milk

processing when compared to static milk processing significantly. In case of static milk

processing (Chapter 4), the maximum volume treated was 48 ml for a 3 min pulsed UV-

light treatment. Despite of increased processing volume, microbial reduction obtained

with continuous milk treatment was comparable to that of static milk treatment.

The effect of flow rate was not statistically significant (p>0.050)). Also there was

an interaction between number of passes and flow rate (p=0.098). In other words, the

log10 reduction at a particular pass also depends on the flow rate. For instance, log10

reductions of 2.07 and 1.36 log10 CFU/ml were achieved at 20 and 40 ml/min,

respectively, when the number of passes were 3 and the distance from the UV-light

source was 8 cm. However, log10 reductions of 7.23 and 0.55 log10 CFU/ml were

obtained at the same conditions when the milk was treated in a single pass.

As indicated earlier, the effect of the number of passes was also investigated to

mimic industrial scale situations where milk may be treated until the desired log10

reduction is achieved to meet the regulatory standards. However, there was no

significant difference (p>0.05) in the number of passes (Figure 5.4 and Figure 5.6).

101

40

log reduction

030

2

4

6

Flow rate5.07.5 2010.0

DistanceHold ValuesPasses 2

Figure 5.5. Surface plot of log10 reduction vs. flow rate and distance.

40

log reduction

030

2

4

6

Flow rate12 20

3Passes

Hold ValuesDistance 8

Figure 5.6. Surface plot of log10 reduction vs. flow rate and distance.

The interaction between number of passes and flow rate was also assessed using

Minitab®. At a single pass treatment, decreased flow rate of the milk increased the log10

reduction. However, when the number of passes were three, there is no significant

change in the log10 reduction when the flow rate is changed.

102

Surface response model

The following surface response model with constant, linear, interactions, and

squared terms was developed using two independent data sets. Variables used in the

model are 1) distance of sample from the UV-light source strobe (D, cm), 2) number of

passes (P), and 3) flow rate (F, ml/min).

Log10 reduction = β0 + β1D+ β2P+ β3F+ β11D2 + β22P2 + β33F2 + β12DP+ β13DF+

β23PF+ ε.

where, ε = Error and β0, β1, β2, β3, β22, β12, β13, and β23 are coefficients (Table 5.3)

Table 5.3. Regression coefficients of response surface model.

Term Coefficient p value β0 -16.21481 0.255 β1 4.94370 0.029* β2 3.68625 0.429 β3 0.05371 0.922 β11 -0.21440 0.053 β22 -1.93708 0.052 β33 -0.00377 0.643 β12 -0.10333 0.691 β13 -0.04017 0.162 β23 0.14925 0.098

*Bold values indicate terms which are statistically significant (p<0.05).

The developed model was validated using an independent data set. The model

predicted data reasonably agree with the experimental data. An R2 of 0.836 (Figure 5.7)

indicates that the model explained 83.6% variation in the data. In an ideal situation, a

perfect fit line will have an R2 of 1 and slope of 1. In this case, the slope was 0.984

which suggest a clear correlation.

103

y = 0.9836xR2 = 0.8359

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Experimental log10 reduction

Pre

dict

ed lo

g 10 r

educ

tion

by R

espo

nse

surfa

ce

met

hod

_ _ Perfect fit line (If predicted = experimental) ___ Trendline passing through origin for the dataset

Figure 5.7. Validation of response surface design.


The temperature of the milk increased gradually during pulsed UV-light

treatment. For instance, the temperature of the milk treated at 8 cm distance from the

quartz window at 30 ml/min flow rate was 1.5, 5.6, and 27.8oC for 1, 60, and 300 sec

treatments, respectively (Figure 5.8). The temperature increases during infrared heat

treatment for single pass milk treatment were given in Figure 5.8. Increase in

temperature because of pulsed UV-light was up to 38oC. Temperature increase might

have a synergistic effect on inactivation of S. aureus in some cases. The initial

temperature of the milk increased as the number of passes increased because of

absorption of thermal energy (Table 5.4). For instance, the final bulk temperature of the

milk was 22, 32, and 36oC, for 1, 2, and 3 passes, respectively, when the milk was treated

at 20 ml/min at 5 cm distance from the quartz window. This temperature build-up might

104

have caused some changes in the quality of the milk. Therefore, it is beneficial to

eliminate the temperature build-up in order to preserve the quality of the food.

Table 5.4. Average bulk temperature of milk after pulsed UV-light treatment.

Distance from quartz window

(cm)

Flow rate (ml/min) 1 pass 2 passes 3 passes

20 22 32 36 30 27 34 37 5 40 24 33 36 20 26 35 39 30 27 35 36 8 40 36 47 51 20 31 37 41 30 30 39 45 11 40 29 39 46

0

5

10

15

20

25

30

35

40

0 50 100 150 200 250 300

Time (sec)

Incr

ease

in te

mpe

ratu

re (o C

)

8cm 20ml/min

8cm 30ml/min 11cm 20ml/min

5cm 20ml/min

11cm 30ml/min

5cm 30ml/min 8cm 40ml/min 11cm 40ml/min

5cm 40ml/min

Figure 5.8. Typical temperature profile during pulsed UV-light treatment of milk.

105

5.5. Conclusions

This study explored the possibility of using pulsed UV-light treatment as an

alternative method for inactivation of S. aureus in milk. In general, pulsed UV-light

effectively inactivated S. aureus in milk, which can be verified by the complete

inactivation obtained in two cases. No growth was observed after enrichment in most

cases. Pulsed UV-light treatment could be an attractive alternative to conventional

pasteurization in terms of energy and cost. It is simple and can deliver lethal doses of

pulsed UV-light for effective inactivation of S. aureus and hence an important technique

in milk processing. The results of this study also suggest that pulsed UV-light treatment

can be potentially applied in commercial scale for continuous milk pasteurization. An

appropriate design of the equipment which will aid better penetration and shorter

treatment time is needed for commercial success. Furthermore, the pulsed UV-light

system can also operate in conjunction with the existing pasteurization system to ensure

the safety of the product. Pulsed UV-light may inactivate pathogens which are resistant

to heat treatment. Further studies have to be done to verify the applicability of pulsed

UV-light treatment in an industrial scale.

5.6. References

Bank, H.L., J.L. Schmehl, and R.J. Dratch. 1990. Bactericidal effectiveness of


Bean, N.H. and P.M. Griffin.1990. Foodborne disease outbreaks in the United States,


Bean, N.H., P.M. Griffin, J.S. Goulding, and C.B. Ivey. 1990. Foodborne disease

outbreaks, 5-year summary, 1983-1987. J. Food Prot. 53: 711-728.

106


Microbiology: The Microbiology of Milk. Vol. 1, R.K. Robinson, ed. New York,




Applied Nutrition – Food and Drug Administration. Available at

http://vm.cfsan.fda.gov/~comm/ ift-puls.html. Accessed 19 April 2006.

Dunn, J.., T. Ott, and W. Clark. 1995. Pulsed light treatment of food and packaging.

Food Technol. 49:95-98.


honey by pulsed UV-light treatment. CIGR J. AE Sci. Res. Dev. Manuscript FP

03-009. Vol. V. 7 pp.


55(12):16.



38:883-888.

McDonald, K.F., R.D. Curry, T.E. Clevenger, K. Unklesbay, A. Eisenstrack, J. golden,


sources for the decontamination of surfaces. IEEE Trans. Plasma Sci. 28(5):1581-

1587.


R.V. Tauxe. 1999. Food-related illnesses and death in the United States. Emerg.

Infect. Dis. 5(5):607-625.

Miller, R., W. Jeffrey, D. Mitchell, and M. Elasri. 1999. Bacterial responses to ultraviolet

light. ASM news. 65(8):534-541.

Olsen S.J., MacKinon L.C., Goulding, J.S., Bean N.H., and Slutsker, L. 2000.


Mortal Wkly. Rep. 49:(SS-1)1-66.

107

http://vm.cfsan.fda.gov/%7Ecomm/%20ift-puls.html




Xenon, 2003. Sterilization and decontamination using high energy light. Woburn, MA:

Xenon Corporation.

108

6. Disinfection of Water by Flow-through Pulsed

Ultraviolet Light Sterilization System*

6.1. Abstract

Disinfection of water is an important task for semiconductor, pharmaceutical,

food or other industries for various purposes. Pulsed ultraviolet light is a novel

technology which offers a rapid and effective solution to achieve sterilization of water

and to provide reductions in the organic load of the water. In this study, efficacy of

pulsed UV-light was studied for inactivation of Bacillus subtilis spores by using a flow-

through, pulsed UV light chamber. Various flow rates up to 14 L/min were evaluated.

The pulsed UV treatment results demonstrated the complete inactivation of B. subtilis for

all the flow rates evaluated, which yielded 5.5 log10 CFU/ml reduction or more.

Furthermore, there was no growth observed after enrichment in either light or no-light

conditions, which indicated that there were no injured cells and no recovery of the spores

because of the photorepair mechanism. Absorption of pulsed UV-light treated water at

254 nm reduced significantly for most of the cases suggesting decrease in the turbidity.

Therefore, this study clearly demonstrated that pulsed UV-light has a potential to be

utilized for sterilization of water.

* This article was presented at Ultrapure water® journal’s “High Purity Water Conference” (Portland, OR, October 25-26, 2005) and accepted for publication in Ultrapure Water® Journal. Copyright held by the Tall Oaks Publishing, Inc., Littleton, CO, USA. Reprinted with permission from Tall Oaks Publishing. The original citation is as follows: Krishnamurthy, K. and A. Demirci. 2006. Disinfection of water through flow-through ultraviolet light disinfection system. Ultrapure Water (submitted).

109

6.2. Introduction

Water is used abundantly in many industries. Water may contain several

pathogenic microorganisms including Vibrio cholerae, Salmonella, Shigella,

Campylobacter, and Escherichia coli O157:H7. Therefore, it is necessary to disinfect the

water before using it for industrial applications to ensure the safety and purity of water.

Ultraviolet (UV) light disinfection is gaining interest among public water systems and

commercial applications to disinfect the drinking water. UV-light does not form harmful

by-products while inactivating pathogenic microorganisms (EPA, 1999). Therefore, UV-

light is viewed as an alternative to chemicals such as chlorine. Continuous UV-light has

been used for disinfection of drinking water since 1906. In addition to the disinfection of

drinking water, UV-light can also be used for disinfection of water used in

semiconductor, pharmaceutical, food, or other industries and for sanitation of wastewater.

UV-light can be applied in two different modes; namely continuous and pulsed

modes. Recently the use of pulsed UV-light has been getting attention since it can

inactivate pathogenic microorganisms in a short period of time with its better penetration

than continuous UV-light. Pulsed light is a broad spectrum of radiation covering UV,

visible, and infrared regions with typical wavelength range from 100 nm to 1100 nm.

Energy is stored in a capacitor and released as very short intermittent pulses (typical

pulse duration ranges from several hundred nanoseconds to microseconds), so the

instantaneous peak energy will be in the order of several Megawatts (McDonald et al.,

2000). However, the total energy is comparable to that of continuous UV-light (in the

order of several watts). Therefore, by using the same amount of total energy, one can

achieve better inactivation using pulsed UV-light because of higher peak energy and

110

constant disturbance caused by pulses. Krishnamurthy et al. (2004; chapter 3) reported

that pulsed UV-light can inactivate Staphylococcus aureus, a foodborne pathogenic

microorganism within several seconds. Within 5 s treatment, up to 8.5 log10 CFU/ml

reduction was achieved. This clearly indicated the effectiveness of pulsed UV-light.

The objective of this study was to investigate the efficacy of the pulsed UV-light

for sterilization of water during continuous water treatment. As spores are more resistant

to UV-light than vegetative cells, Bacillus subtilis spores were used in the study to

evaluate the efficacy of the pulsed UV-light system in a worst case scenario.


Microorganism

Bacillus subtilis (ATCC 6633) was obtained from American Type Culture

Collection (Manassas, VA) and kept as frozen culture at -80oC. The culture was

transferred to 150 ml of Tryptic soy broth (TSB, Difco, Sparks, MD) and grown for 24 h

at 37oC and then transferred to tryptic soy agar (TSA) slants. After incubating at 37oC for

24 h, the slants were stored in the refrigerator until further use. Sub-culturing was

performed on TSA slants every other week in order to ensure the culture viability.

Spore preparation

Four different methods of spore preparation were tested by changing the growth

media, wash buffer, and/or number of days of incubation in order to maximize the spore

count. Using the culture stored on TSA slants at 4oC, streak plating was done on TSA

followed by incubation at 37oC for 24 h. A single colony from the plate was transferred

111

to 10 ml of TSB and incubated. All the incubation was done at 37oC for 24 h unless

noted.

Method 1: The prepared culture was spread on TSA plates and incubated for 3

days. Then, the plates were rinsed with 5 ml of KCl/0.5 M NaCl solution and disturbed

gently with a sterile spreader to remove the spores from the plates. Rinsing was repeated

with another 5 ml of KCl/0.5 M NaCl solution and the rinse solution was transferred to

sterile centrifuge bottles. The solution was vortexed in order to maintain the homogeneity

followed by centrifugation at 3,800 x g, at 4oC for 10 min (Sorvall Super T 21, ST-H750,

Kendro Lab Products, Newton, CN), After centrifugation, the cells were washed with

250 ml of 950 mM Tris-HCl/EDTA buffer and re-centrifuged. Washing with 250 ml of

950 mM Tris-HCl/EDTA buffer was repeated two more times. The washed cells were

resuspened in phosphate buffer (supplemented with Tween 20, pH 7.4, Sigma-Aldrich,

St. Louis, MO) and the cells were heat shocked to produce spores at 80oC for 10 min. The

spore suspension was stored at 4oC until further use. It was believed that the high amount

of Tris-HCl and EDTA in the buffer resulted in injury to the cell leading to very low final

spore concentration. Therefore, a lower concentration of Tris-HCl and EDTA were used

in method 3.

Method 2: The prepared culture was grown in TSB for 7 days at 37oC. The

sample was centrifuged at 3,800 x g at 4oC. After centrifugation, the cells were washed

with 250 ml of phosphate saline buffer and re-centrifuged. Washing with 250 ml of

phosphate saline buffer was repeated two more times. The washed cells were resuspened

in phosphate saline buffer and the cells were heat shocked at 80oC for 10 min to produce

spores. The spore suspension was stored at 4oC until further use.

112

Method 3: The prepared culture was spread on TSA plates and incubated for 7

days at 37oC. The plates were rinsed with 5 ml of KCl/0.5 M NaCl solution and disturbed

gently with a sterile spreader. In order to remove the spores from the plates, rinsing was

repeated with another 5 ml of KCl/0.5 M NaCl solution and the rinse solution was

transferred to sterile centrifuge bottles. The solution was vortexed to maintain

homogeneity and followed by centrifugation at 3,800 x g at 4oC for 10 min. After

centrifugation, the cells were washed with 250 ml of 10 mM Tris-HCl/EDTA buffer and

re-centrifuged. Washing with 250 ml of 10 mM Tris-HCl/EDTA buffer was repeated two

more times. The washed cells were resuspened in phosphate saline buffer and the cells

were heat shocked to produce spores at 80oC for 10 min. The spore suspension was stored

at 4oC until further use.

Method 4: The prepared culture was spread on TSA plates (50 plates/batch) and

incubated for 7 days at 37oC. The plates were rinsed with 5 ml of KCl/0.5 M NaCl

solution and disturbed gently with a sterile spreader to remove the spores from the plates.

Rinsing was repeated with another 5 ml of KCl/0.5 M NaCl solution and the rinse

solution was transferred to sterile centrifuge bottles. The solution was vortexed to

maintain the homogeneity and followed by centrifugation at 3,800 x g at 4oC for 10 min.

After centrifugation, the cells were washed with 250 ml of phosphate saline buffer and

re-centrifuged. Washing with 250 ml of phosphate saline buffer was repeated two more

times. The washed cells were resuspened in phosphate saline buffer and the cells were

heat shocked to produce spores at 80oC for 10 min. The spore suspension was stored at

4oC until further use.

113

Pulsed UV-light treatment system

Pulsed UV-light treatment was done with a SteriPulse®-RS 4000 pulsed light

sterilization system (Xenon Corporation, Wilmington, MA) (Figure 6.1). The system

generated 1.27 J/cm2/pulse of radiant energy at 1.8 cm below the quartz window surface

of the UV-lamp and produced polychromatic radiation in the wavelength range of 100 to

1100 nm, with 54% of the energy being in the UV-light region. The system produced 3

pulses of 360 µs duration per second.

Figure 6.1. Pulsed UV-light sterilization system.

The system chamber had an annular cylinder arrangement with an UV lamp being

placed at the center (Figure 6.1). The water disinfection system was made up of stainless

steel and had 10.2 cm outer diameter and 40.6 cm length. A site glass was provided to

facilitate the observation of water flow and to measure the UV-light intensity.

114

Figure 6.2. Schematic diagram of cross section of the Steripulse®-RS 4000 chamber.

Water Inlet

UV lamp

Quartz glass separation

Annular space for test

Water outlet

Polished wall

The annular space between the UV-lamp and the outer wall of the vessel were

separated by a quartz sleeve to enhance the transmission of UV-light to water. The

maximum volume of water in the disinfection chamber at any given time was 2.9 L (i.d.

of the outer vessel was 9.8 cm, o.d. of the inner quartz tube was 2.5 cm, length of the

chamber was 40.6 cm, and added volume because of the site glass was 0.05 L). The water

was moved with a centrifugal pump (TE5-5C-MD, Emerson motor company, St. Louis,

MD), and the flow rate was adjusted by a control valve. The water from a 10 or 20 L

carboy container (Cole-Parmer, Vernon Hills, IL) was pumped through the water vessel.

The flow of the water was measured and adjusted using a flow meter (F-41017L, Blue

white industries, Huntington Beach, CA).

115

Cleaning of the flow-through pulsed UV-light system

In order to avoid any cross contamination in the pulsed UV system, pulsed UV-

light was coupled with chlorine solution followed by sterile D.I. water rinse to remove

any chlorine residues after several different combinations were investigated to get a

sterile system. The final cleaning procedure was determined as follows: 1) circulate 10 L

sterile deionized (D.I.) water with pulsed UV-light on for 10 L/min for 10 min for 10 L of

water and 30 min for 20 L of water, 2) circulate 10 L of 200 ppm chlorine solution for 10

min, 3) circulate 10 L of sterile D.I. water for 10 min, 4) pump sterile D.I. water to adjust

the target flow rate.


D.I. water was autoclaved at 121oC for 60 min and cooled overnight at room

temperature. Ten ml of the prepared spore suspension for 10 L of D.I. water or 20 ml of

the prepared spore suspension for 20 L of D.I. water was added and mixed well to ensure

homogeneity. The initial spore population in water determined by plating on TSA was

5.5-6.5 log10 CFU/ml. The UV-light system was activated, and the inoculated water was

pumped through the system at the set flow rates of 2, 4, 6, 8, 10, and 14 L/min, which

yielded 88, 44, 29, 22, 18, and 13 s residence times in the chamber, respectively. After

50% of the water was passed through, about one liter of sample was collected. The pulsed

UV-treated water was analyzed for microbial reduction by plating on TSA followed by

incubation at 37oC. Two replications were performed for each treatment. Enrichment was

performed for all treatments by transferring 1 ml of pulsed-UV treated water into 9 ml of

TSB and incubating at 37oC for 24 h. Enrichment was performed to ensure that there

116

were no injured cells. In order to find out that the cells would be recovered by repairing

the UV damage under light exposure because of the photorepair mechanism, enrichment

was also performed under light.

Turbidity measurement

The absorption of the untreated and the treated samples were measured at 254 nm

using a UV-Vis spectrophotometer (DU series 500, Beckman, Fullerton, CA) to monitor

the turbidity of the water.

Radiant energy measurement

UV-light energy absorbed by the water was measured by using a radiometer

(Ophir PE50, Ophir Optronics Inc., Wilmington, MA) by measuring the broadband UV-

light intensity for each pulse by placing the pyroelectric detection head on the quartz

window provided for light measurements on the water treatment chamber. The

radiometer was calibrated at 254 nm. The UV-light intensity at 254 nm was determined

by comparing the UV-light intensity obtained using another radiometer

(SED240/ACT5/W detector head, International lights, Newburyport, MA) as per the data

given by Xenon Corporation (Wilmington, MA.)


The bulk temperature of the water before and after treatment was measured by

placing a thermometer at the center of the container after mixing it well. Several

measurements were taken and average was reported.

117


Method used for spore harvesting played a significant role in getting a higher

spore concentration (Table 6.1). Spores grown on agar medium yielded more spores than

broth as thin agar plates provide less nutrition over 7 days of incubation. Also greater

number of days of incubation resulted in a higher spore concentration as more vegetative

cells produce spores because of lack of nutrition and moisture over the period of

incubation.

Table 6.1. Evaluation and comparison of spore harvesting methods.

Final spore

concentration (log10 CFU/ml)

Method # Spore preparation method

950 mM Tris-Hcl/EDTA buffer + 3 days incubation on Tryptic soy agar 1 1.45

Phosphate buffer saline procedure + 7 days incubation in Tryptic soy broth 2 4.33

10 mM Tris-Hcl/EDTA buffer + 7 days incubation on tryptic soy agar 3 5.43

Phosphate buffer saline procedure + 7 days incubation on tryptic soy agar 4 8.34

The wash solution also played a significant role in getting higher spore

concentration as some wash solutions might have injured the spores resulting in lower

spore counts. Since spore suspension prepared by method 4 yielded the highest spore

concentration (8.34 log10 CFU/ml), Method 4 was followed to prepare the inoculum.

The pulsed UV treatment results demonstrated the complete inactivation of B. subtilis for

all the flow rates evaluated (2, 4, 6, 8, 10, and 14 L/min), which yielded 5.5 log10

reduction or more (Table 6.2). Initial inoculum concentration for each flow rate was

slightly different (ranged from 5.5 to 6.5 log10 CFU/ml). Furthermore, there was no

118

growth observed after enrichment in dark or under light, which indicated that there were

no injured cells and no recovery of the spores because of the photorepair mechanism.

Therefore, all the spores subjected to pulsed UV-light treatment were completely

inactivated under the evaluated conditions.

Table 6.2. Inactivation of Bacillus subtilis spores by pulsed UV-light treatment.

Growth after enrichment*

Flow rate (L/min)

Population (Log10 CFU/ml)

0 (control) 5.5 – 6.5 Yes 2 0 No 4 0 No 6 0 No 8 0 No 10 0 No 14 0 No

*Both under light and no-light conditions.

Absorption at 254 nm of pulsed UV-light treated water was reduced significantly

for most of the cases suggesting reduction in turbidity (Table 6.3), which suggested that

pulsed UV-light treatment not only disinfects the water, but also disintegrates the organic

material by oxidation which results in purer sterile water.

Table 6.3. UV-light absorption at 254 nm.

UV-light absorption at 254 nm Flow rate

(L/min) Untreated Pulsed UV treated 2 0.093±0.023 -0.285±0.054 4 0.000±0.093* -0.146±0.113 6 0.131±0.076 -0.055±0.064 8 0.136±0.042 -0.061±0.073 10 0.052±0.039 -0.095±0.042 14 0.028±0.069 0.064±0.073

*Negative value replaced with 0.00.

119

Broadband energy per pulse (J/pulse), power (W), power per area (W/cm2), and

estimated power at 254 nm (W/cm2) were reported for each tested flow rate on Table 6.4.

The energy delivered by pulsed UV-light was measured using the radiometer suggesting

that approximately 0.21 W/cm2 (difference between the broadband power with no water

(0.56 W/cm2) and average broadband power with water (0.35 W/cm2)) of the energy is

absorbed by inoculated water (Table 6.4). Some portion of the UV-light was absorbed

directly by the microorganism and some by water resulting in an increase in the bulk

temperature, up to 6oC under the evaluated conditions.

Table 6.4. UV-light energy measurements during pulsed UV-light treatment1.

Broadband energy per

pulse (J/pulse)

Estimated power at 254 nm4 (W/cm2)

Bulk temperature increase (oC)

Broadband power per

area3 (W/cm2)

Broadband power2

(W) Flow rate

0 L/min (No

water) 3.35 10.05 0.56 0.0112 N/A

2 L/min 2.13 6.39 0.35 0.0070 2 4 L/min 2.12 6.36 0.35 0.0070 3 6 L/min 2.12 6.36 0.35 0.0070 3 8 L/min 2.11 6.33 0.35 0.0070 4 10 L/min 2.10 6.30 0.35 0.0070 5 14 L/min 2.04 6.12 0.34 0.0068 6

1The Radiometer was calibrated at 254 nm. Broadband energy is reported throughout. 2Three pulses were produced per second. 3Radiometer had a 48 mm diameter area of exposure. 4Based on the comparison of measurements with SED240/ACT5/W radiometer detector head, as suggested by Xenon Corporation, 2% of the broadband energy was at 254 nm based on the comparison studies.

120

6.5. Conclusions

Pulsed UV-light treatment has shown to be very effective in inactivating B.

subtilis spores in this study. The results clearly show the potential of pulsed UV-light to

be utilized for water disinfection cost-effectively. Testing at higher flow rates is needed

for the optimization of the system. In general, vegetative cells need less energy than

spores to be inactivated, and hence pulsed UV-light can be used effectively to inactivate

pathogens in a short period of time with less energy. Especially pulsed UV-light can

inactivate Cryptosporidium parvum, a protozoa of major concern in water, effectively as

it is less resistant than Bacillus subtilis spores. Boeger et al. (1999) reported that one

pulse of pulsed UV-light inactivated 1.00 and 4.60 log10 CFU/ml of Bacillus subtilis and

Cryptosporidium parvum, respectively. Therefore, pulsed UV-light has a potential to be

utilizied for disinfection of vegetative cells, bacterial spores, and protozoa such as

Cryptosporidium parvum. Also a pulsed UV-light provides a mercury free UV-light

treatment which does not produce any hazardous by-products and environmentally

friendly.

6.6. References



http://www.epa.gov/safewater/mdbp/pdf/alter/chapt_8.pdf. Accessed 5 March

2006.

McDonald, K.F, R.D. Curry, T.E. Clevenger, K. Unklesbay, A. Eisenstrack, J. Golden,


sources for the decontamination of surfaces. IEEE Trans. Plas. Sci. 28: 1581-

1587.

121


Fine, F. and P. Gervais. 2004. Efficiency of pulsed UV light for microbial

decontamination of food powders. J. Food Prot. 67:787-792.

Boeger, J.M., W.H. Cover, and C.J. McDonald. 1999. PureBright sterilization system:

Advanced technology for rapid sterilization of pharmaceutical products, medical

devices, packaging and water. Wiesbaden, Germany: Proceedings of Hypro99

congress.

122

7. Infrared Heat Treatment for Inactivation of

Staphylococcus aureus in Milk

7.1. Abstract

The efficacy of infrared heating for inactivation of Staphylococcus aureus, a

pathogenic microorganism, in milk was studied to investigate the potential of this

technology for milk pasteurization. S. aureus population was reduced from 0.10 to 8.41

log10 CFU/ml, depending upon the treatment conditions. The effects of infrared lamp

temperature (536, 619oC), volume of the treated milk sample (3, 5, and 7 ml), and

treatment time (1, 2, and 4 min) were found to be statistically significant (p<0.05).

Complete inactivation of S. aureus was obtained in two cases within 4 min at a 619oC

lamp temperature, resulting in 8.41 log10 CFU/ml reduction. Enrichment resulted in

growth as some of the injured cells were able to repair. Further investigation of infrared

heat treatment for longer treatment times (> 4 min) indicated that there was no growth

observed following enrichment in most cases for treatment at a 619oC lamp temperature.

The results demonstrated that infrared heating has an excellent potential for effective

inactivation of S. aureus in milk. Further optimization of the process may result in a

commercially successful milk pasteurization method.

123

7.2. Introduction


between 0.5 and 1000 μm (Rosenthal, 1996), which is mainly utilized for processing food

materials. Far infrared radiation has been used in the industry since the 1950s in United

States (Skjoldebrand, 2001) because of the following advantages: 1) higher heat transfer

capacity, 2) instant heating because of direct heat penetration, 3) high energy efficiency,

4) faster heat treatment, 5) fast regulation response, 6) better process control, 7) no

heating of surrounding air, 8) equipment compactness, 9) uniform heating, 10)

preservation of vitamins, and 11) less chance of flavor losses from burning of foods

(Dagerskog and Osterstrom, 1979; Afzal et al., 1997; Skjoldebrand, 2002).

Recently, there is an increasing interest in the applicability of infrared radiation

for inactivation of pathogens. Food components and microorganisms absorb effectively

in far-infrared region (3 to 1000 μm), resulting in heating of food systems and thus

inactivation of pathogens. Infrared heating inactivates the pathogen by damaging

intracellular components such as DNA, RNA, ribosome, cell envelope, and/or proteins in

the cell (Sawai et al., 1995). Absorption of infrared energy by water molecule in

microorganisms is one of the important factors for microbial inactivation since water

absorbs readily in the infrared region and results in rapid temperature increase

(Hamanaka et al., 2006).

Bacterial inactivation is influenced by the peak wavelength of the lamp,

temperature of the lamp, physical state of the microorganism, depth of the food,

composition, shape and surface characteristics of food, etc. Hamanaka et al. (2006)

treated B. subtilis spores on stainless steel Petri dishes with varying water activities using

124

three infrared lamps having peak wavelength of 950, 1100, and 1150 nm. Water activity

played a vital role in the inactivation of bacterial spores since the state and amount of

water molecule present in the spores affect the effective absorption of infrared energy.

The spores treated with 950, 1100, and 1150 nm peak wavelength radiation were most

resistant to water activities of 0.9, 0.7, and 0.6, respectively, resulting in maximum D

values of 5.7, 21.3, and 32 min, respectively.

Escherichia coli O157:H7 suspended in 0.05 M physiological phosphate buffered

saline was treated with infrared radiation at radiative power of 0.322 J/s/cm2 on the

surface of bacterial suspension (Sawai et al., 1995). The temperature of the bacterial

suspension increased exponentially from approximately 280oK to 336oK during a 10 min

infrared irradiation treatment. The inactivation effect of infrared heating was better than

convective heating for the same heating time and same bulk temperature rise in medium

(Sawai et al., 1995). E. coli cells were inactivated even when the cell suspension was

cooled down, where the bulk temperature was below lethal level. Although the effect of

bulk temperature was negligible, because of the absorption of infrared energy in a thin

layer of the bacterial suspension solution, inactivation of E. coli occurred. The

temperature of the thin film at the surface was much higher than that of bulk temperature.

Consequently, the authors suggested that differences in inactivation effects were because

of this increased temperature in the thin film in case of infrared heating. They also noted

that sensitivity to rifampicin was increased followed by increase in sensitivity of

cholaramphenicol during both infrared and convective heating, suggesting that

inactivation occurred mainly because of damage to RNA polymerase and ribosome

(Sawai et al., 1995).

125

Giraffa and Bossi (1984) investigated the efficacy of infrared heating on

microbial reduction in milk. Infrared heat treatment of milk at 65oC and 1000 L/h flow

rate resulted in reductions of standard plate count, Psychotropic bacteria, Coliforms,

Enterococci, Lactic acid bacteria (grown in MRS agar), and Lactic acid bacteria (grown

in M17 agar), by 63%, 96% 99.9%, 80%, 71%, and 60%, respectively. The surface

pasteurization effect of infrared radiation in cottage cheese was investigated by Rosenthal

et al. (1996). Yeast and mold cells were reduced by approximately 3 log10 CFU/ml when

treated with an infrared lamp with energy 250 J/s at a distance of 2.5 to 3 cm from the

cheese surface. Even after 8 weeks of storage at 4oC, less than 100 yeast and mold

cells/g was found, suggesting that infrared pasteurization is effective for surface

pasteurization.

Hashimoto et al. (1992a) obtained reductions of more than 4.5 log10 CFU/ml of S.

aureus and 2 log10 CFU/ml of E. coli 745 with an irradiation power of 7.5x10-7 J/s-cm2

when using selective agar. The FIR effect on the pasteurization of bacteria on or within

wet-solid medium was evaluated by Hasimoto et al. (1992b). A complete inactivation of

E. coli was obtained with irradiation at 4.36x10-1 J/s-cm2 for 6 min., which corresponds

to approximately 2 log10 CFU/plate when there is no medium added on top of the

bacteria. However, when the medium was added on top of the plated bacteria to a

thickness of 1-2 mm, approximately 1 log10 CFU/plate was obtained under the same

experimental condition.

Hashimoto et al. (1993) investigated the effect of irradiation power on IR

pasteurization below lethal temperature of bacteria. Approximately 4 log10 CFU/ml and

1 log10 CFU/ml reductions of S. aureus were obtained by FIR and NIR, respectively,

126

when the irradiation power was 7.57x 10-1 J/s-cm2, indicating that the FIR heating is

more effective in inactivating the microbial population than NIR heating.

Jun and Irudayaraj (2003) studied the effect of selective infrared heating on

inactivation of Aspergillus niger and Fusarium proliferatum in corn meal. The spectral

wavelength was manipulated using a bandpass filter (5.45 to 12.23 μm). Approximately

2 log10CFU/g reduction of Aspergillus niger was obtained with 5 min treatment time with

or without bandpass filter. Reductions of 1.5 and 2 log10 CFU/g of Fusarium

proliferatum were obtained for 5 min with and without bandpass filter, respectively. The

log10 reduction obtained for both Aspergillus nigher and Fusarium proliferatum with

filter were slightly higher than that obtained without a filter.

If the food is treated with high power infrared radiation for a short period of time,

surface sterilization of food material can be achieved. As infrared heating mainly heats a

thin layer from the surface, the food product can be rapidly cooled after infrared

treatment and thus provides less change in the quality of food material because of

negligible heat conduction (Hamanaka et al., 2000). Surface sterilization of wheat for

inactivation of natural spoilage microorganisms was investigated by Hamanaka et al.

(2000). Reductions of 0.83, 1.14, 1.18, and 1.90 log10 CFU/g were obtained with 60 s

treatment with 0.5, 1, 1.5, and 2 kW infrared heaters, respectively.

Infrared heating has lower energy requirements to achieve the same temperature,

leading to lower operational costs. Hebber et al. (2004) reported that the specific energy

consumption for drying of potato were 17.1 and 7.60 MJ/kg of water, for hot air drying

and infrared drying, respectively. The specific energy consumptions for drying of carrot

were 16.15 and 7.15 MJ/kg of water for hot air drying and infrared drying, respectively.

127

This clearly shows that energy consumptions were reduced to 44% and 38% for potato

and carrot drying. Furthermore, infrared heating also resulted in reduction of the total

processing time for drying. Therefore, infrared heating has a potential to be utilized

instead of conventional heating. The efficacy of infrared heating for inactivation of

Staphylococcus aureus, a pathogenic microorganism, in milk was studied to investigate

the potential of this technology for milk pasteurization.



Staphylococcus aureus (ATCC 25923; Penn State Food Microbiology Culture

Collection, University Park, PA) cells were grown in 150 ml Tryptic Soy Broth (TSB;

Difco, Sparks, MD) at 37oC for 24 h, and harvested by centrifugation (Sorvall STH750,

Kendro Lab Products, Newtown, CT) at 3,300 x g for 25 min at 4oC. A 100 ml sample of

raw milk (obtained from the University Creamery or dairy farm at the Pennsylvania State

University, University Park, PA) was added to the centrifuged S. aureus cell pellet from

previous step to yield approximately 8 to 9 log10 CFU/ml.

Infrared heating system

A lab scale, custom-made infrared heating system with a cone-shaped, aluminum

waveguide (35 cm diameter at top, 2.5 cm diameter at bottom, and 44 cm high) was used

in this study (Figure 7.1 and Figure 7.2). The waveguide was made up of aluminum

because of its high emissivity of 0.8.

128

Stand

Solid state relays

Power supply

Parallel port

Temperature recorder

Wave guide

IR lamps

Milk sample

Thermocouple

Figure 7.1. Schematics of the infrared heating system.

Figure 7.2. Lab scale Infrared heating system.

129

The system and the control program were originally developed by Jun and

Irudayaraj (2003) and the existing system was modified to suit the current application.

The infrared heating system had six ceramic infrared lamps (Mor Electric Heating

Association, Inc., Comstock Park, MI). The infrared lamp is a half trough emitter with a

maximum power of 500 W for 120 V input and had a cast-in K type thermocouple. All

the lamps were fixed inside the closing on top of the waveguide and arranged symmetric

to the central axis of the waveguide. The system was controlled by a digital controller

(PC) via a program written using Lab Windows software (ver 4.01, National Instruments,

Austin, TX). The control program written in Lab Windows is given in Appendix C. The

lamp temperature was continuously measured using K-type thermocouples through a data

acquisition system (model 34970A; Hewlett-Packard Co., Englewood, CO) via a 20-

channel amplifer/multiplexer and A/D converter. Based on the temperature reading and

the control logic used in the software, digital signals were sent through the parallel port to

turn on/off the infrared lamps through a solid state relay, in order to maintain the

temperature set. The Graphical User Interface (GUI) was used to establish the

temperature/wavelength setting and to turn the system on/off.

Experimental design

A full factorial design with three factors and 2-3 levels was utilized in this study.

The factors were average temperature of the infrared lamps (536, 619oC), volume of the

milk sample (3, 5, and 7 ml), and treatment time (1, 2, and 4 min). Three replications

were performed for each condition. Two different temperatures were obtained by

allowing the lamps to reach steady state temperature at two conditions. For the first

130

condition, the program was used to control the temperature of the lamp using solid state

relays, and the lamps were allowed to reach a steady state temperature.

The average temperature of the infrared lamps was 536±20oC. In the second

condition, the lamps were directly connected to an 120 V power supply rather than

controlling the lamp temperature by manipulation. In this way, the lamps reached their

maximum temperatures. The average temperature of the infrared lamps was 619±5oC.

Also, the effect of longer treatment times (> 4 min) was investigated by treating the

microorganisms for 5, 10, and 15 min in addition to the treatments up to 4 min.


Milk, artificially inoculated with Staphylococcus aureus cells was treated with

infrared heat by placing the sample approximately 1 cm below the bottom opening of the

waveguide. The milk sample was kept in an autoclaved polypropylene dish with

approximately 3.3 cm diameter and 1.7 cm height.


Untreated and infrared heat treated milk samples were analyzed for

Staphylococcus aureus by serially diluting and plating on Baird-Paker agar base with a

spiral plater (Autoplate® 4000; Spiral Biotech, Norwood, MA), followed by incubation at

37oC for 24 h. Finally, surviving fractions of S. aureus were enumerated by Q-CountTM

(Spiral Biotech). The log10 reduction of S. aureus population was calculated by

estimating the difference between average log10 value of control samples and infrared

heat treated samples. Furthermore, an enrichment procedure was performed for zero or

131

low counts by transferring 1-ml of sample into 9-ml of TSB and incubated at 37oC for 24-

48 h.


The temperature of the milk during infrared heat treatment was monitored using

K-type thermocouples attached to a data-logger (Omegaette HH306, Omega Engineering

Inc., Stamford, CT).


The obtained data were analyzed statistically by ANOVA (Analysis of Variance -

general linear model) using MINITAB® (version 13.3; Minitab Inc., State College, PA).

A full quadratic model with all the interactions was used for the data analysis with a 95%

confidence level. Extreme outliers were removed from the statistical analysis in order to

reduce the bias. Because of the variation in log10 reductions of replications, sometimes it

is possible to get negative reduction for mild treatments. However, theoretically, there

can not be a negative reduction and hence it was replaced with zero reduction.


Infrared heating effectively inactivated S. aureus in milk within four minutes.

Reductions of 0.10 to 8.41 log10 CFU/ml was obtained (Table 7.1). Complete

inactivation of S. aureus was obtained at 619oC lamp temperature within 4 min, when the

sample volume was 3 or 5 ml. However, positive growth during enrichment indicated

that some of the cells were injured because of infrared heating and were able to repair.

132

Table 7.1. Log10 reduction values of S. aureus in milk by infrared heat treatment.

Log10 reduction1,2 1 min 2 min 4 min Vol

ume 536oC 619oC 536oC 619oC 536oC 619oC A 3.43 ± 0.89 B,a

A 2.96 ± 1.043 AB,a

A 8.41 ± 0.004 C,b

A 0.10 ± 0.17 A,a

A 0.29 ± 0.21 A,a

A 1.21 ± 0.003 A,a 3 ml

A 0.16 ± 0.27 A,a

A 0.18 ± 0.28A,a

A 0.27 ± 0.47 A,a

B 1.37 ± 0.62 A,a

A 2.96 ± 0.96 B,a

A 8.41 ± 0.004 B,b 5 ml

A 0.16 ± 0.28 A,a

A 0.14 ± 0.24 A,a

A 0.27 ± 0.47 A,a

B 0.57 ± 0.37 A,a

A 1.66 ± 0.80A,a

B 3.45 ± 1.853 B,a 7 ml

1Average of three replications is listed with the standard deviation (outliers were not used for calculation of average). Initial inoculum was 8.41 ± 0.09 log10 CFU/ml. 2Values not preceded by the same upper case letter in the same column are significantly different from each other. Values not followed by the same upper case letter for the specific temperature (536 or 619oC) in the same row are significantly different from each other for that particular temperature. Values not followed by the same lower case letter for the specific time level (1, 2, or 4 min) in the same row are significantly different from each other for that particular time level. 3Extreme outliers were not included for data analysis. 4There was growth observed after enrichment indicating injured cells.

All of the main effects (temperature, volume of sample, and treatment time) were

statistically significant (p<0.05) (Table 7.2 and Figure 7.3). All the interactions

(time*volume, volume*temperature, and temperature*time interactions in analysis of

variance) were also statistically significant (p<0.05) (Table 7.2 and Figure 7.4). An R2 of

0.967% indicates that the analysis of variance was able to explain about 97% of the

variation in the data.

133

Mea

n of

log

redu

ctio

n

753

4

3

2

1

0619536

421

4

3

2

1

0

Volume (ml) Temperature (C)

Time (min)

( ) g

Figure 7.3. Main effects plot (fitted means) for log10 reduction

Table 7.2. Analysis of variance for log10 reduction of S. aureus.

Degrees

of freedom

Sequential sum of squares

Adjusted sum of squares

Adjusted mean square

F value Source P value

Volume 2 34.08 22.77 11.38 30.74 0.000* Temperature 1 38.49 38.91 38.91 105.05 0.000

Time 2 197.58 176.72 88.36 238.56 0.000 Volume*Temperature 2 7.66 7.92 3.96 10.68 0.000

Temperature*Time 2 43.35 37.59 18.80 50.75 0.000 Volume*Time 4 20.65 21.50 5.37 14.51 0.000

Volume*Temperature *Time 4 6.28 6.28 1.57 4.24 0.007

Error 32 11.85 11.85 0.37 Total 49 359.36

*Bolded values indicate statistically significant terms (p<0.05).

134

Volume (ml)

5.0

2.5

0.0

T emperature (C)

T ime (min)

421

619536

5.0

2.5

0.0

753

5.0

2.5

0.0

357

(ml)Volume

536619

Temperature (C)

124

(min)Time

Figure 7.4. Interaction plot (fitted means) for log10 reduction.

Effect of treatment time

The effect of treatment time on inactivation of S. aureus was significant (p<0.05)

(Table 7.1). As expected, increase in the lamp temperature resulted in increased log10

reduction because of increased energy absorption and temperature increase. For instance,

reductions of 0.29, 3.43, and 8.41 log10 CFU/ml were obtained at 619oC lamp

temperature for 1, 2, and 4 min treatment time, respectively, when 3 ml of milk was

treated. The rate of inactivation of S. aureus was increased rapidly after a 2 min

treatment, indicating that the temperature of the milk sample reached the lethal level. As

there was growth observed after enrichment for 4 min infrared treatment at 619oC, the

effect of longer treatment times was investigated by treating S. aureus at the specified

conditions for up to 15 min (Table 7.3). Complete inactivation of S. aureus was obtained

in all the tested conditions, corresponding to 8.41 log10 CFU/ml. The enrichment

135

procedure performed indicated that for treatments longer than 5 min at 619oC lamp

temperature there was no growth observed in most cases. However, infrared treatments

at a lamp temperature of 536oC resulted in growth after enrichment exhibiting that some

of the cells were injured and able to repair themselves.

Table 7.3. Infrared heat treatment of S. aureus for longer time.

Growth after enrichment

5 min 10 min 15 min 536oC 619oC 536oC 619oC 536oC 619oC

3 ml Yes No Yes No No No 5 ml No Yes No No Yes No 7 ml Yes No Yes No Yes No

The interaction of treatment time with volume and temperature (time*volume and

time*temperature terms in the analysis of variance) were statistically significant (p<0.05),

indicating that there was a close correlation. For instance, lower sample volume and

lower temperatures resulted in lower reductions, while the large volume and higher

temperature combinations resulted in larger inactivation (Figure 7.4).

Effect of lamp temperature

Inactivation of S. aureus obtained at 536 and 619oC were statistically significant

(p<0.05) (Figure 7.3 and Table 7.2). For instance, reductions of 2.96, 2.96, and 1.66

log10 CFU/ml were obtained for treatment of 3, 5, 7 ml milk samples at 4 min at 536oC,

while reductions of 8.41, 8.41, 3.45 log10 CFU/ml were obtained at 619oC lamp

temperature for a 4 min treatment. Generally lower temperatures in combination with

shorter treatment times and lesser volume resulted in lower reductions as evident from

the significance of temperature*volume and temperature*time interactions (p<0.05).

136

Also it is noted that 619oC lamp temperature was very effective in inactivation of S.

aureus as compared to 536oC. Increase in lamp temperature resulted in increase in

available infrared energy for microbial inactivation. The temperature of the milk sample

increased rapidly during the infrared heat treatment (Figure 7.5).

0

20

40

60

80

100

120

140

0 50 100 150 200 250 300 350

Time (sec)

Tem

pera

ture

(o C)

Relay 3 ml TopDirect 3 ml BottomDirect 5 ml TopRelay 5 ml TopDirect 5 ml BottomDirect 3 ml TopDirect 7 ml TopRelay 3 ml BottomRelay 5 ml BottomRelay 7 ml TopDirect 7 ml BottomRelay 7 ml Bottom

Figure 7.5. Temperature increase during infrared heating.

As seen in Figure 7.5, the temperature of the milk increased significantly higher

when there was no relay as the infrared lamp temperature was higher. As expected the

rate of temperature increase was higher when lesser volume of milk was treated as more

energy is readily available to heat the milk sample. The temperature was raised up to 55

to 105oC within five minutes of infrared heat treatment. Optimizing the temperature of

milk during infrared heat treatment could result in less detrimental quality changes.

137

Effect of volume of milk

Infrared radiation mainly heats a thin layer of milk sample from the surface,

because of its poor penetration capacity. Therefore, it is vital to know the effect of

volume on inactivation of S. aureus. In general, an increase in the sample milk volume

resulted in lower inactivation as infrared radiation can not penetrate deep and heats up

only a few millimeters below the surface of the milk sample. Reductions of 3.43, 1.37,

and 0.57 log10 CFU/ml were obtained when 3, 5, 7 ml volume of milk were treated at

619oC for 4 min, respectively (p<0.05).

7.5. Conclusions

Complete inactivation of S. aureus was obtained in two cases within 4 min at

619oC. The corresponding inactivation was 8.41 log10 CFU/ml. However, the

enrichment was positive indicating there was cell injury. Further inactivation studies at

longer treatment time indicated that, most of the samples treated for more than 5 min at

both 536 and 619oC resulted in no detectable colonies. However, few treatments were

positive after enrichment procedure indicating that cells were injured because of infrared

heat treatment. As expected, S. aureus treated at 536oC was able to grow during

enrichment. In case of treatments performed at 619oC, most of the enrichment was

negative indicating that higher temperature resulted in complete inactivation of S. aurues

with no cell injury. This shows that infrared heating has a potential to be utilized for

microbial inactivation. The effects of volume, treatment time, and lamp temperature and

their interactions were significant (p<0.05). Generally, lower volume of milk, longer

treatment time, and higher lamp temperature resulted in greater inactivation of S. aureus.

138

Infrared heating requires less energy and reduces the treatment time when compared to

conventional heating (Afzal et al., 1999). Spectral manipulation of infrared radiation

results in selective heating of food components. Proper spectral manipulation of infrared

radiation might result in selective heating of S. aureus in milk without heating other food

components and result in fewer quality changes in milk as compared to conventional

heating and yet produce milk which is safe to consume.

Further studies on optimization of the process, sensory evaluation, and quality

changes during infrared heating have to be investigated in detail. Optimization of the

infrared heating process to maintain the quality of milk may result in an alternative

heating method for milk pasteurization.

7.6. References

Afzal, T.M., T. Abe, and Y. Hikida. 1999. Energy and quality aspects of combined FIR-

convection drying of barley. J. Food Eng. 42:177-182.

Dagerskog, M. and L. Osterstrom. 1979. Infrared radiation for food processing I: A study

of the fundamental properties of infrared radiation. Lebensm.-Wiss. Technol.

12(4):237-242.

Hamanaka, D., S. Dokan, E. Yasunga, S. Kuroki, T. Uchino, and K. Akimoto. 2000. The

sterilization effect of infrared ray on the agricultural products spoilage

microorganisms. ASABE paper no. 006090. St. Joseph, MI: American Society of

Agricultural and Biological Engineering.

Hamanaka, D., T. Uchino, N. Furuse, W. Han, and S. Tanaka. 2006. Effect of the

wavelength of infrared heaters on the inactivation of bacterial spores at various

water activities. Int. J. Food Microbiol. 108:281-285.

Hashimoto, A., J. Sawai, H. Igarashi, and M. Shimizu. 1992a. Effect of far-infrared

irradiation on pasteurization of bacteria suspended in liquid medium below lethal

temperature. J. Chem. Eng. Jap. 25:275-281.

139

Hashimoto, A., H. Igarashi, and M. Shimizu. 1992b. Far-infrared irradiation effect on

pasteurization of bacteria on or within wet-solid medium. J. Chem. Eng. Jap.

25:666-671.

Hashimoto, A., J. Sawai, H. Igarashi, and M. Shimizu. 1993. Irradiation power effect on

IR pasteurization below lethal temperature of bacteria. J. Chem. Eng. Jap. 26:331-

333.

Hebber, H.U., K.H. Vishwanathan, and M.N. Ramesh. 2004. Development of combined

infrared and hot air dryer for vegetables. J. Food Engg. 65:557-563.

Jun, S. and J. Irudayaraj. 2003. A dynamic fungal inactivation approach using selective

infrared heating. Trans. ASAE. 46(5):1407-1412.

Rosenthal, I., B. Rosen, and S. Bernstein. 1996. Surface pasteurization of cottage cheese.

Milchiwissenschaft. 51(4):196-201.

Sawai, J., K. Sagara, H. Igarashi, A. Hashimoto, T. Kokugan, and M. Shimizu. 1995.

Injury of Escherichia coli in physiological phosphate buffered saline induced by

far-infrared irradiation. J. Chem. Engg. Jap. 28(3):294-299.

Skjoldebrand, C. 2001. Infrared heating. In Thermal Technologies in Food Processing. P.

Richardson, ed. New York, NY: CRC Press.

Skjoldebrand, C., S.V.D. Hark, H. Janstad, and C.G. Andersson. 1994. Radiative and

convective heat transfer when baking dough products. University of Bath, United

Kingdom: Proceedings of 4th Bath Food Engineering Conference.

140

8. Microscopic and Spectroscopic Analysis of

Inactivation of Staphylococcus aureus by Pulsed UV-

Light and Infrared Heating.

8.1. Abstract

Pulsed UV-light and infrared heat treated S. aureus cells were analyzed using

transmission electron microscopy to identify the damages caused during the treatment. A

five second treatment of S. aureus with pulsed UV-light resulted in complete inactivation

of S. aureus even after enrichment. The temperature increase during the pulsed UV-light

treatment was 2oC. S. aureus was treated using six ceramic infrared lamps with the

power of 500 W. A 5 ml of S. aureus cells in phosphate buffer was treated at 700oC lamp

temperature for 20 min. The microscopic observation clearly indicated that there was cell

wall damage, cytoplasmic membrane shrinkage, cellular content leakage, and mesosome

disintegration for both pulsed UV-light and infrared treatments. The structural damage of

S. aureus during pulsed UV-light treatment might be caused by the constant disturbances

of the intermittent pulses. Temperature increase might be the cause of the cellular

damage by infrared heat treatment. FTIR microspectrometry was successfully used to

classify the pulsed UV-light and infrared heat treated S. aureus by discriminant analysis.

Further investigation on identification of key absorption bands may result in a better

assessment of the chemical and structural changes during pulsed UV-light and infrared

heating.

141

8.2. Introduction

Pulsed UV-light is produced by accumulating the energy in a capacitor and

releasing it as a short duration pulse to magnify the power greatly. There is an increased

interest in using pulsed UV-light for inactivation of pathogenic microorganisms in recent

years because of the very short period of time required. Pulsed UV-light is a broad-band

spectrum in the wavelength range of 100-1100 nm, with approximately 54% of energy in

the ultraviolet range.


between 0.5 and 1000 μm (Rosenthal, 1996). Far-infrared radiation can be used for

heating of food systems and inactivation of pathogens because of higher absorption of

energy in the far-infrared wavelength range (3 to 1000 μm) by microorganism and food

components. Therefore, infrared heating has a potential to be used for microbial

inactivation in foods.

It is important to know the underlying mechanism of microbial inactivation to

optimize the inactivation process. Transmission electron microscopy (TEM) and infrared

spectroscopy can be utilized for this purpose. TEM is a high resolution microscope,

which uses an electron beam to discriminate cellular level details of the microorganisms.

TEM can distinguish points closer than 0.5 nm (Prescott et al., 1999). A thin section of

bacterial cell is treated with various chemicals to stabilize the cell structure, cut into a

thin slice using a diamond or glass knife, stained to increase the contrast, and exposed to

electron beam. As the composition of the cell components differ, the intensity of electron

scattering also varies thereby producing an image of the internal structure of the bacteria.

Several researchers have used electron microscopy techniques successfully for the

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investigation of inactivation mechanisms of several novel technologies such as pulsed

electric field, antimicrobial agents, etc. (Brouillette et al., 2004; Calderon-Miranda et al.,

1999; Liu et al., 2004). The effect of pulsed electric field and nisin on Listeria innocua in

skim milk was investigated using TEM by Calderon-Miranda et al. (1999). They noticed

several features of pulsed electric field damaged cells such as lack of cytoplasm, cell wall

damage, cytoplasmic clumping, increase in cell membrane thickness, poration of cell

wall, and leaching of cellular content. Ruptures of cell wall and cell membranes were

observed at selected electric field intensities. Liu et al. (2004) explained that the

inactivation of bacteria by chitosan is because of cell wall damage by using TEM. The

outer membrane of chitosan treated E. coli was altered after chitosan treatment. The cell

membrane of chitosan treated Staphylococcus aureus was disrupted and the cellular

contents were leaked.

Infrared spectroscopy is a chemical analytical method which can be used to

determine the chemical and structural information of the target material based on

vibration transitions. Various food/microbial components absorb infrared light at specific

wavelengths. Thus, infrared spectroscopy produces a fingerprint of spectral absorption

characteristics of the biological components by providing absorption/transmission

characteristics over time. The spectrum obtained is transformed from the time domain

into frequency domain by Fourier transformation, so that absorption with respect to a

particular wavelength could be assessed (Wilson and Goodfellow, 1994).

Fourier transform spectroscopy (FTIR) can be used to discriminate pathogenic

microorganisms by spatially resolving the structural and compositional information of the

microorganisms at molecular level (Yu and Irudayaraj, 2005; Gupta et al., 2004). Liu et

143

al. (2004) utilized FTIR for determination of the interaction between chitosan and

synthetic phospholipids membrane in an effort to understand the basic inactivation

mechanism. Several researchers successfully utilized FTIR for the discrimination of

microorganisms and to investigate the changes in chemical composition because of

several processes. Therefore, the use of TEM and FTIR will be beneficial for the

investigation of inactivation mechanisms for pulsed UV-light and infrared treatments.


Inoculum preparation

Staphylococcus aureus (ATCC 25923; Penn State Food microbiology culture

collection, University Park, PA) was grown at 37oC for 24 h, followed by centrifugation

at 3,300 x g for 25 min. The cells were resuspended in 0.1 M phosphate buffer (pH 7.2;

Becton Dickinson microbiology systems, Sparks, MD) to yield about 8-9 log10 CFU/ml.


Phosphate buffer artificially inoculated with S. aureus was treated with pulsed

UV-light (Steripulse-XL 3000® pulsed light sterilization system, Xenon Corporation,

Wilmington, MA). A 12-ml sample of S. aureus kept in an aluminum dish of 7-cm

diameter was treated at 8-cm below the quartz window of the sterilization system. The

distance between the central axis of the UV-strobe and the quartz window was 5.8-cm. S,

aureus were treated for 1, 2, 4, 8, 16, and 32 s for spectroscopic analysis and 5 s for

microscopic analysis.

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A lab scale, custom-made infrared heating system developed by Jun and

Irudayaraj (2003) was used for infrared heat treatment. The infrared heating system

comprised of six ceramic infrared lamps and the temperature was controlled by a Lab

Windows program (ver 4.01, National Instruments, Austin, TX) using solid state relays.

The lamps had a maximum power of 500 W for 120 V input. The bacterial sample was

treated approximately cm below the infrared lamps. A 5 ml of S. aureus cells in

phosphate buffer was treated at 700oC for 1, 2, 4, 8, and 16 min for spectroscopic

analysis. A 5 ml of phosphate buffer with S. aureus were treated at 700oC for 20 min to

evaluate the structural damage during infrared heat treatment using microscopic analysis.

TEM analysis

Pulsed UV-light and infrared heat treated S. aureus cells were further processed

for the TEM imaging as follows:

Fixation and embedding of bacterial cells: Steps for this process were as

follows: 1) Primary fixation: In order to inactivate the cells, to stop the enzyme activity,

and to crosslink proteins, the cells were treated overnight with 2.5% glutaraldehyde in 0.1

M sodium cacodylate solution at 4oC; 2) Buffer wash: The aldehyde residues were

removed by washing the cells with 0.1M sodium cacodylate for five minutes. This

procedure was repeated three times; 3) Secondary fixation: Secondary fixation was

performed with 1% osmium tetroxide in 0.1M sodium cacodylate buffer in order to

provide electron density for contrast while TEM imaging by oxidizing the double bonds

in unsaturated fatty acids and forming monoesters; 4) Buffer wash: To remove excess

145

osmium, a buffer wash was performed three times for five minutes each; 5) En bloc

staining: Buffer washed cells were stained in filtered 2% Uranyl acetate in a dark

container for 1 h. This procedure further preserves the membranes and provides electron

density for TEM imaging; 6) Dehydration: A dehydration procedure was performed to

replace air spaces in the tissues. The samples were washed for 10 min in each of the

following solutions: 50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol (3 times),

100% EM grade ethanol (3 times), and 100% acetone (3 times). Initially the air spaces

were replaced by increasing concentrations of ethanol and then ethanol was replaced with

acetone; 7) Infiltration: The dehydrated samples were treated in a rotor with graded series

of increasing concentrations of resin and acetone until the concentration of resin was

100%. The samples were treated for 2 h each in the following solutions: 50:50 acetone:

resin, 25:75 acetone: resin, and 100% resin (3 times); and 8) Embedding and

polymerization: The specimen in the resin was heated overnight at 60oC to polymerize

the resin and to form a solid block of bacterial cells.

Sectioning and staining: Excess resin was trimmed off to expose and facilitate

easy access to bacterial cells during sectioning. Ultra-thin sections (~30 nm thickness) of

bacteria were sliced using a diamond knife (Ultra-45; Diatome, Fort Washington, PA) in

an Ultramicrotome (Reichard-Jung, Vienna, Austria). The sections were collected in a

grid. A staining procedure was performed as follows; 1) The grids were immersed with

the section-side down in filtered 2% Uranyl acetate in 50% ethanol for 16 min, 2) The

grids were then immersed quickly in nanopure water (Carbon dioxide removed by boiling

for 5 min) and vertically agitate gently for 1 min, 3)Excess water on the grids were

removed and they were dried completely, 4) In a staining dish, grids were immersed for

146

12 min section side down in filtered lead stain (mixture of 1200 µl boiled nanopure water

with no carbon dioxide, 4.65 mg lead citrate, and 11.85 µl 10 N sodium hydroxide) for,

5) Grids were placed in a beaker with 40 ml of boiled water and two drops of 10 N

sodium hydroxide solution and washed by vertically agitating for 30 s, 6) Grids were

placed next in a beaker with 40 ml of boiled water and one drop of 10 N sodium

hydroxide solution and washed by vertically agitating for 30 s, 7) Grids were placed then

in a beaker with 40 ml of boiled water for 1 min, and 8) Finally excess water was

removed and the grid were dried completely.

TEM imaging: Stained ultra-thin sections of bacterial cells were imaged using

transmission electron microscopy (JEM 1200 EXII; JEOL, Peabody, MA). The specimen

(grids with sections of bacterial cells) was placed in a specimen rod and inserted in the

TEM. The height of the specimen was adjusted. The brightness of the electron beam

was adjusted and the bacterial cells were focused clearly. Images of the sections of

bacterial cells were taken at different magnifications using a high resolution camera

(F224; Tietz, Gauting, Germany).

FTIR spectroscopy analysis

S. aureus cells treated with pulsed UV-light or infrared heating were stored at 4oC

until other samples were treated, all samples were further processed as described below.

Cell wall and cytoplasm were separated from treated/control S. aureus cells. The whole

cells and cell walls were evaluated with FTIR spectroscopy as follows:

Sample preparation for spectroscopic evaluations: 1) Whole cell: One

milliliter samples of un-treated and treated S. aureus cells were transferred into sterile 1.5

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ml micro-centrifuge tubes (VWR International, West Chester, PA) and processed in a

mini-centrifuge (Model no. C-1200, National Labnet Corporation, Woodbridge, NJ) for 5

min at 1,000 rpm. The supernatant was decanted and the cell pellet was smeared onto a

gold coated glass slide (~200-nm thickness of gold) and dried for 24 h in a dessicator at

room temperature; 2) Cell wall: The cell wall of S. aureus was separated by treating cells

in a sonicator (Branson 1510 sonicator) at 40 kHz for 10 min. Ultrasonicated cells were

centrifuged at 30,000 x g for 30 min to separate the cytoplasm and cell wall. The cell

wall was deposited at the bottom of the centrifuge as a pellet and the cytoplasm was

suspended in the supernatant. Cell walls were then smeared onto a gold slide and dried

as explained above.

FTIR measurements: Mid infrared spectra of the bacterial cell and cell wall

were measured using a Digilab Excalibur FTS 6000 spectrometer fitted with a UMA 600

infrared microscope (Digilab, Randolph, MA). A ceramic air-cooled IR source with a

kBr beam was used. The mercury-cadmium-telluride detector was cooled with liquid

nitrogen during data collection. The sample chamber was purged with helium to

minimize the interference from water vapor. Three measurements were taken at different

spots from each replication; hence a total of 9 spectra were collected for each treatment.

Averages of 128 scans were collected for each spectrum from 600 to 6000 cm-1 spectral

region at a resolution of 4 cm-1.

Discriminant Analysis: Using Partial Least Square (PLS) and Canonical Variate

Analysis (CVA), the raw data were conditioned by baseline correction and area

normalization to reduce the bias. First and second derivative methods were utilized for

spectral analysis to detect the amide I and amide II protein regions. The data were

148

analyzed using PLS and CVA as follows: i) Partial Least Square (PLS): PLS is a well

established evaluation technique and used widely for the identification and classification

of data. It decomposes the original matrix into several products of multiplication

corresponding to the concentration, loadings and scores that indicate the variation of the

data as well as the degree of fit. In this study, PLS was performed in the spectral range

between 800-3000 cm-1. The spectral data were subjected to PLS data compression.

Then, each spectrum was reconstructed by a linear combination of the product of scores

and their weights (loading). The resulting calculations were used for multiple group

classification; 2) Canonical Variate Analysis (CVA): The second method used for

discriminating between groups of observations was the Canonical Variate Analysis.

Canonical variate scores have successively maximized dofferences between group

variances and within groups variances, and the CV loadings have been obtained as

eigenvectors of a matrix given by

[W–1] [B] (Equation 1)

where, W was the within–groups covariance matrix, and B was the between–groups

covariance matrix. The objective of this procedure was to minimize the within–group

variance and to maximize the between-groups variance. The goodness of fit was

indicated by the % correct classification. Discriminant models were developed based on

the calibration data and evaluated separately using the validation data set. The correctly

classified samples were expressed as a percentage of the total number of samples in the

specific groups. The spectra were normalized by dividing the intensity values

corresponding to each wavenumber in the spectrum by its standard deviation before

analysis. All the data were converted in a PLS algorithm for classification and

149

identification after Fourier smoothing of the spectra using GRAMS-32 software. Using

800-3000 cm-1, it was able to classify the time course bacteria treated with IR and UV

radiation.


Transmission electron microscopy

The effect of pulsed UV-light and infrared heat treatment on S. aureus was

investigated using Transmission Electron Microscopy (TEM). Selected images of

damages induced by pulsed UV-light and infrared heating have been shown in Figure 8.1

to Figure 8.3.

Microscopic analyses of S. aureus cells indicate that there were damaged cells

occurring both during infrared heating (Figure 8.1A) and pulsed UV-light treatments

(Figure 8.1B). In the case of pulsed UV-light, cells were treated for only 5 s. However,

the results were comparable to those of infrared heat treatment for 20 min at 700oC lamp

temperature.

Pulsed UV-light treatment: Pulsed UV-light treated S. aures cells exhibited

severe damage though the cells were treated for only 5 s with pulsed UV-light. Figure

8.2B indicates cell wall damage and cell content leakage at several locations. Thus, some

cells lacked cell wall because of the disintegration of cell wall (Figure 8.2C).

Furthermore, it was evident that the cytoplasmic membranes were shrinking and the

internal cellular structures were collapsing.

150

A B

C

Figure 8.1. Comparison of damages observed by TEM: A) Control sample, B) Infrared heat treated sample, and C) Pulsed UV-light treated sample.

Cytoplasmic membrane shrinkage results in the loss of semi permeability of the

membrane and hence the osmotic equilibrium of the cell is disturbed. This leads to

leakage of cellular content from the cytoplasm and cell death. As mentioned earlier, the

samples were treated with pulsed UV-light for just five seconds and there were no

significant temperature increases during the treatment. No growth was observed after a 5

s treatment with pulsed UV-light even after enrichment, indicating that S. aureus was

completely inactivated.

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A B

C D

Figure 8.2. Evaluation of pulsed UV-light induced damages in S. aureus by TEM: A) Control sample, B) Cell wall rupture, C) Lack of cell wall, D) Cytoplasm shrinkage and

cell wall damage, E) Cytoplasm shrinkage and membrane damage, and F) Cell wall damage and cellular content leakage.

E F

152

Traditionally, it is believed that the inactivation mechanism for pulsed UV-light

was mainly because of thymine dimer formation in the bacterial cell. However,

microscopic observation indicated that damage to cellular structure occurred with pulsed

UV-light treatment. Therefore, it was evident that some other mechanism could

contribute to the inactivation of pulsed UV-light in addition to thymine dimer formation.

It was evident that some S. aureus cells were inactivated by thymine dimer formation

since a majority of the pulsed UV-light treated cells were intact without any structural

damage. This fact indicated that the cells might have been inactivated by thymine dimer

formation because microbiological studies indicated that there was no growth and all the

cells were completely inactivated.

It is clear that the temperature of the sample increased rapidly during pulsed UV-

light treatment (chapter 3). Therefore, some researchers suggested that pulsed UV-light

may also have a thermal effect on the bacteria (Fine and Gervais, 2004; Takeshita et al.,

2003; Wekhof, 2000). Because of the difference in absorption of pulsed light energy by

bacteria and surrounding media, vaporization of water in the bacteria occured, leading to

a small steam flow in the bacterial cells causing cell disruption. Though the thermal

effect plays a vital role at longer treatments with pulsed UV-light (>5 s treatment time), it

is negligible for shorter treatments. In this study, S. aureus cells were inactivated for 5 s,

and there was no significant temperature increase observed. Therefore, the damage

occurred to S. aureus by pulsed UV-light in this study was not attributed to thermal

damage.

The photochemical transformation, thymine dimer formation, did not damage the

cellular structure of the bacteria and there was no thermal damage under the tested

153

conditions. Therefore, there should be some other effect than photochemical or photo-

thermal. The inactivation mechanisms observed in this study were similar to those of

cells treated with pulsed electric field (Barbosa-Canovas et al., 1999; Calderon-Miranda

et al., 1999; Dutreux et al., 2000). Thus, it can be hypothesized that cellular damage

could be because of the pulsing effect. In pulsed UV-light, the energy is stored in a

capacitor and released as intermittent pulses with high energy in several MW ranges.

The pulse duration ranges from several nano seconds to micro seconds, and there are

several pulses produced per second. The pulsed UV-light system utilized in this study

produced 3 pulses/s with pulse duration of 360 µs. Because of constant disturbance

caused by the pulses, the bacterial cells may undergo stress and hence result in structural

damage. Therefore, the inactivation mechanisms of pulsed UV-light can be divided into

the following:

a. Photochemical effect: The inactivation is mainly caused by chemical changes in

the DNA and RNA. Thymine dimer formation is the major photochemical

change attributed to microbial inactivation. There may also be other minor

chemical bond formations and/or breakages in bacteria.

b. Photothermal effect: There is a significant temperature increase during longer

duration pulsed UV-light treatment. As the heating rate of the bacterial cell and

surrounding media are different, localized heating of bacteria occur, which leads

to cell death.

c. Photophysical effect: Because of constant disturbance caused by the high

energy pulses, structural damages to bacteria may occur. Therefore, the

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effectiveness of pulsed UV-light treatment can be improved by optimizing the

pulse width and number of pulses.

These results clearly indicate that the inactivation mechanism of pulsed UV-light

is different from that of continuous UV-light. Several researchers have suggested that

pulsed UV-light is up to four times more effective in inactivation of microorganisms.

This study suggests that this increased effectiveness can be attributed to photophysical

and photothermal effects of pulsed UV-light.

Infrared heat treatment: The cell wall, cytoplasm, and mesosome of the

untreated S. aureus cells were intact as seen in Figure 8.3A. However, these structures

were damaged in infrared heat treated S. aureus cells (Figure 8.3). The microscopic

observations indicate that there was cell wall damage leading to absence of cell wall

(Figure 8.3B). This image clearly indicated that the cell wall was not present for the

infrared heat treated sample. Cell wall damage lead to leakage of cellular content

including genetic material (Figure 8.3C). It was also noted that the cytoplasmic

membrane shrunk upon infrared heat treatment, and damage to the cytoplamic membrane

was also observed (Figure 8.3D and Figure 8.3F). The internal cellular structure, the

mesosome was also damaged during infrared heat treatment (Figure 8.3E). The control

sample had an intact mesosome in the cytoplasm (Figure 8.3A).

155

A B

C D

EFigure 8.3. Microscopic evaluation of damages to S. aureus because of infrared heat treatment: A) Control sample, B) Lack of cell wall,

C) Cell wall breakage and cytoplasm content leakage, D) Cytoplasm shrinkage, E) breakage in mesosome, and F) Cytoplasm

damage.

F

156

FTIR spectroscopy

The pulsed UV-light treated and infrared heat treated cells and cell walls of S.

aureus were successfully classified by using FTIR spectroscopy. Discriminant analyses

of the spectroscopic data are given in Figure 8.4 to Figure 8.6. Distinct clusters were

formed for different treatments and treatment times indicating that the FTIR absorption

spectra of individual treatments were different. Thus, the treatment time and treatment

had a significant impact on the changes in chemical and/or structural changes in the cells.

In general, pulsed UV-light treated cells had a better discriminate power than infrared

heat treated cells. Also, the characteristics of the spectra for the whole cells and cell

walls were significantly different. Hence, the discriminant analysis was able to

differentiate this.

Since the composition of cell walls and whole cells differ significantly,

differences in the spectra are expected. In this study, cell walls were also used because,

during pulsed UV-light and infrared heating, cell wall damage was noticed. A cell wall is

made up of polysachharides and proteins, therefore one can expect their contribution to

be prominent in the FTIR spectra (Chenxu and Irudayaraj, 2005). However, DNA and

RNA are the major contributors for the cytoplasm extract (Chenxu and Irudayaraj, 2005)

and hence, in the whole cell, one can expect the contributions from DNA, RNA, proteins,

polysachharides. The data are in agreement with this observation. The result shows that

the data from the whole cell and cell wall were classified in different clusters (Figure 8.6)

for both pulsed UV-light and infrared heat treatments. The infrared heat treated whole

cells were spread out indicating that there was significant difference within the infrared

heat treated samples at different treatment times.

157

-8

-6

-4

-2

0

2

4

6

8

10

-8 -6 -4 -2 0 2 4 6 8 10

CV1

CV

2

32 sec 16 sec 8 sec 4 sec 2 sec 1 sec 0 sec (Control) A

-10

-8

-6

-4

-2

0

2

4

6

8

-6 -4 -2 0 2 4 6 8

CV1

CV

2

10

32 sec 16 sec 8 sec 4 sec 2 sec 1 sec 0 sec (Control)B

Figure 8.4. Classification of pulsed UV-light treated Staphylococcus aurues by PLS-CVA (PLS Factor- 4): A) whole cell, B) cell wall.

158

-6

-4

-2

0

2

4

6

8

10

-10 -8 -6 -4 -2 0 2 4 6 8

CV1

CV2

16 min 8 min 4 min 2 min 1 min 0 min (Control) A

-8

-6

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0

2

4

6

8

-10 -5 0 5 10

CV1

CV2

16 min 8 min 4 min 2 min 1 min 0 min (Control)

B Figure 8.5. Classification of infrared heat treated Staphylococcus aurues by PLS-CVA

(PLS Factor- 4): A) whole cell, B) cell wall.

159

-50

-40

-30

-20

-10

0

10

20

30

-40 -20 0 20 40 60 8

Score 1

Sco

re 2 0

Cell wall Whole Cell A

-15

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-5

0

5

10

15

20

-35 -30 -25 -20 -15 -10 -5 0 5 10 15

Score 2

Sco

re 4

Cell wall Whole cellB

Figure 8.6. Differentiation of cells and cell walls of Staphylococcus aurues by PLS technique: A) pulsed UV-light treated cells, B) Infrared heat treated cells.

160

As FTIR was able to differentiate cells treated for different times, it can be

utilized for rapid measurement of dose received. FTIR measurements may provide an

easier and faster way to determine the adequacy of the pulsed UV-light or infrared heat

treatment by classification of the spectral information. For commercial success of an

emerging technology, it is crucial to have a rapid measurement technique for the

verification of the effective dose absorbed by the food material. For instance, the

effectiveness of pasteurization is tested by testing the activity of the enzyme alkaline

phosphatase. The results showed that FTIR was successfully able to differentiate S.

aureus treated for different time and thus different dosage. Thus, FTIR may be used to

validate the adequacy of the pulsed UV-light and infrared heat treatment.

Further detailed studies have to be done for the assignment of absorption bands to

investigate the chemical and structural changes during pulsed UV-light or infrared

heating. The tentative assignment of absorption bands in the infrared region of microbial

cells is given in Table 8.1.

161

Table 8.1. Absorption bands of microbial cells in the infrared region*

Possible biomolecule contributors

Wavenumber (cm-1) Functional group assignment Reference

2957 γ (CH3) asymmetric Fatty acids Mordechai et al., 2000 2919 γ (CH2) asymmetric Fatty acids Mordechai et al., 2000 2872 γ (CH3) symmetric Fatty acids Naumann, 2000 2852 γ (CH2) symmetric Fatty acids Naumann, 2000

1790-1750 γ (C=O) affected by Cl, etc Not clear Socrates, 2001 1741 γ (C=O) Lipid esters Naumann, 2000

Mordechai et al., 2000; Naumann, 2000 1708 γ (C=O), H-bonded RNA, DNA

Amide I band components resulting from anti parallel plated sheets and β turns

Proteins Naumann, 2000 ~1695

~1670 γ (C==N) RNA/DNA bases Socrates, 2001 ~1655 Amide I of α-helical structure Proteins Naumann, 2000 ~1637 Amide I of β-sheets Proteins Naumann, 2000 1548 Amide II Proteins Naumann, 2000 1515 Shoulder Proteins Naumann, 2000 1457 δ (CH2) Lipids, Proteins Mordechai et al., 2000

Carbohydrates, DNA/RNA

backbone, proteins 1415 C-O-H in plane bending Socrates, 2001

Lipids, carbohydrates,

proteins 1402 δ C(CH3)2 symmetric Lucassen et al., 1998

1312 Amide III Proteins Lucassen et al., 1998 1284 Amide III Proteins Lucassen et al., 1998 1240 (P=O) asymmetric Phospholipids Lucassen et al., 1998

DNA and RNA backbone

Naumann, 2000; Lucassen et al., 1998 ~1160 δ (COP), (CC), (COH)

(CC) skeletal trans conformation

DNA and RNA backbone

Mordechai et al., 2000; Lucassen et al., 1998 ~1120

C-O-C, C-O dominated by ring vibrations 1200-1000 Carbohydrates Naumann, 2000

DNA and RNA, phospholipids

Naumann, 2000; Lucassen et al., 1998 1085 γ (P==O) symmetric

γ (CC) skeletal cis conformation

DNA and RNA backbones

Mordechai et al., 2000; Lucassen et al., 1998 1076

C==C, C==N, C-H in ring structure 900-800 Nucleotides Socrates, 2001

δ – Deformation and γ - Stretch *Yu and Irudayaraj, 2005.

162

Possible assignment of absorbance bands to the pulsed UV-light and infrared

heat-treated cells may result in identification of chemical and structural changes in S.

aureus cells during the pulsed UV-light and infrared heat treatment. Yu and Irudayaraj

(2005) reported that because of overlapping absorbance of a multitude of cellular

compounds in the infrared range, the bands are not highly resolved and difficult to apply.

For assignment of absorption bands, only the cells which were treated for the longest

time were used to observe the maximum changes in the whole cells. Therefore, S. aureus

cells treated with infrared heat for 16-min or treated with pulsed UV-light for 32-s, were

used for the assignment of absorption bands.

Pulsed UV-light treated cells has a strong absorption around 1100 cm-1 indicating

that contribution from 1085 cm-1 (DNA, RNA, phospholipids) and 1076 cm-1 (DNA and

RNA backbones) were there. Microscopic observations indicated that there were cell

wall damage and cellular content leakage, even after 5 sec treatment (Figure 8.2). Since,

the pulsed UV-light sample was treated for 16 s, one can expect more damage to the cell

wall and cell lysis. This resulted in leakage of DNA, RNA, and other internal content of

the cytoplasm. Therefore, there was a strong absorption in the RNA/DNA range.

Absorption by carbohydrates (1200 to 1000 cm-1) might also have contributed to the

strong absorption at 1100 cm-1 (Figure 8.7). Pulsed UV-light treated cells also had an

absorption band at 1160 cm-1, corresponding to DNA and RNA backbone absorption,

indicating that there was significant leakage of genetic material from the treated cells.

Furthermore, absorption in the protein band was also observed, especially at 1284, 1312,

and 1548 cm-1. This clearly suggested significant cell wall damage during pulsed UV-

light treatment.

163

-10.00

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000

Wavenumber (cm-1)

Abs

orpt

ion

Control 32 sec

Possible finger print areas for pulsed UV-light treated whole cells (32 sec):

DNA and RNA backbones: 1160, and 1120 cm-1

DNA, RNA, phospholipids:1085, and 1076 cm-1

Carbohydrates 1200-1000 cm-1

Protein (Amide III): 1284,1312, and 1548 cm-1

Figure 8.7. Assignment of absorption bands for pulsed UV-light treated S. aureus.

Unlike, pulsed UV-light treatment, infrared heat treated S. aureus cells had

significant absorption at possible fatty acids bands of 2957, 2919, 2872, and 2852 cm-1

(Figure 8.8). Furthermore, bands because of possible lipid ester absorption at 1741 cm-1

could be also noted. Infrared heat treated cells had very strong absorption at 1708 (RNA,

DNA), 1402 (lipids, carbohydrates, proteins), and 1120 cm-1 (DNA and RNA

backbones). These absorptions strongly suggest that the infrared heat treated cells were

damaged significantly and the cellular contents leaked. This was verified with TEM

pictures, wherein cell wall and cytoplasmic membrane damage were observed (Figure

8.1). Absorption at the above bands indicated that there was cytoplasmic cell membrane

damage and leakage of the cellular contents.

164

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000

Wavenumber (cm-1)

Abs

orpt

ion

Control 16 min

Possible finger print areas for Infrared heat treated whole cells (16 min): Fatty acids: 2957, 2919, 2872, and 2852 cm-1

Lipid esters: 1741 cm-1

DNA and RNA backbones: 1120 cm-1

Lipids, carbohydrates, and proteins:1402 cm-1

RNA and DNA: 1708 cm-1

Protein (Amide I): 1695 cm-1

Figure 8.8. Assignment of absorption bands for infrared heat treated S. aureus.

8.5. Conclusions

The microscopic observations indicated a severe cellular damage after a 5-s

pulsed UV-light treatment. There was no significant temperature increase during this

treatment indicating that the damage might be because of the pulse effect. Cell wall

damage, cytoplasmic membrane shrinkage, cell content leakage, mesosome rupture, and

genetic material leakage are some of the phenomenon observed by microscopy.

Similarly, infrared heat treated samples also exhibited cell wall damage, condensation of

cytoplasm, and cellular content leakage. Results indicated that these emerging

technologies have a potential to be used for inactivation of pathogenic microorganisms.

165

Further detailed investigation on possible inactivation mechanism may result in some

useful information.

FTIR was used to classify the pulsed UV-light and infrared heat treated S. aureus

cells successfully. Therefore, FTIR has a potential to be used as a rapid assessment tool

for determination of sufficient dosage absorbed by the microbial cells, since the treatment

times correspond to the dosage received during the treatment. Tentative absorption bands

were assigned for the FTIR spectral data. These band assignments, strongly suggested

that there was damage to cell walls, cytoplasmic membranes and leakage of cellular

content. This was in agreement with the microscopic observations. Further detailed

studies could result in successful band assignment and information on the chemical and

structural changes in S. aureus cells induced by pulsed UV-light or infrared heat

treatment.

8.6. References

Barbosa-Canovas, G.V., M.M. Gongora-Nieto, U.R. Pothamamury, and B.G. Swanson.

1999. Preservation of Foods with Pulsed Electric Fields. New York, NY:

Academic Press.

Calderon-Miranda, M.L., G.V. Barbosa-Canovas, and B.G. Swanson. 1999. Transmission

electron microscopy of Listeria innocua treated by pulsed electric fields and nisin

in skimmed milk. Int. J. Food Microbiol. 51:31-38.

Dutreux, N., S. Notermans, T. Wijtzes, M.W. Gongora-Nieto, G.V. Barbosa-Canovas,

and B.G. Swanson. 2000. Pulsed electric fields inactivation of attached and free-

living Escherichia coli and Listeria innocua under several condiditons. Int. J.

Food Microbiol. 54:91-98.


decontamination of food powders. J. Food Prot. 67(4):787-792.

166

Gupta, M.J., J. Irudayaraj, and C. Debroy. 2004. Spectroscopic quantification of bacteria

using artificial neural networks. J. Food Prot. 67:2550-2554.

Lucasssen, G.W., P.J. Caspers, and G.J. Puppels. 1998. In vivo infrared and Raman

spectroscopy of human stratum corneum. Proc. of SPIE.3257:52-61.

Mordechai, S., A. Salman, S. Argov, B. Cohen, V. Erukhimovitch, J. Goldstein, O.

Chaims, and Z. Hammody. 2000. Fourier transform infrared spectroscopy of

human cancerous and normal intestine. Proceedings of SPIE. 3918:66-77.

Naumann, D. 2000. Infrared spectroscopy in microbiology. In Encyclopedia of Analytical

Chemistry. Meyers, R.A. ed. 102-131. Chichester, United Kingdom: John Wiley

& Sons. Ltd.

Socrates, G. 2001. Infrared and Raman characteristic group frequencies. Tables and

Charts. New York, NY: John Wiley and Sons.

Wilson, R.H. and B.J. Goodfellow. 1992. Mid-infrared spectroscopy. In Spectroscopic

Techniques for Food Analysis. Wilson, R.H., ed. New York, NY: VCH

publishers.

Yu, C. and J. Irudayaraj. 2005. Spectroscopic characterization of microorganisms by

Fourier transform infrared microspectroscopy. Biopolymers. 77:368-377.

167

9. Conclusions and Scope for Future Research

Increased consumer awareness about minimally processed foods and industries’

thirst to reduce the total cost of food processing, propel researchers to investigate

alternative food processing technologies. This research was a step in the direction of

identifying two novel food processing technologies - namely pulsed UV-light and

infrared heating - as potential food processing technologies, especially for microbial

decontamination. Consumption of food contaminated with pathogenic microorganisms

cause illnesses and deaths resulting in several billion dollars loss. Because of rigorous

Governmental regulations and potential risk of costly recalls, food industries are forced to

ensure that their food products are free from pathogenic microorganisms.

The main focus of this study was to investigate the efficacy of pulsed UV-light on

inactivation of Staphylococcus aureus and Bacillus subtilis. The effects of pulsed UV-

light on inactivation of S. aureus on an agar surface (to represent solid food) and a 0.1M

phosphate buffer (to represent liquid food) were investigated. Complete inactivation of S.

aureus on agar seeded cells was achieved within 5 s of pulsed UV-light treatment

resulting in 8.5log10 CFU/ml. Reductions of 7.5 log10 CFU/ml were achieved at 1, 2, and

5 s treatment of S. aureus in a 0.1M phosphate buffer, when the volume of buffer was 12,

24, and 48 ml, respectively. As the volume of the sample increases, the inactivation

decreases because of poor penetration of UV-light. There was no significant temperature

increase during the pulsed UV-light treatment indicating that inactivation was achieved

by pulsed UV-light alone. Following an enrichment procedure, no growth was observed,

indicating that there were no injured cells. These results indicated that pulsed UV-light

has a potential to be used for surface sterilization of food products and to treat liquid

168

foods with high transparency. UV-light, however has poor penetration capacity. Pulsed

UV-light might have better penetration capacity than continuous UV-light. However,

only a small portion of the germicidal UV-light might be available for inactivation of

pathogenic microorganisms as several other components may absorb UV-light and other

radiation energy. Furthermore, the food components may provide protection for the

microorganisms. Hence, it is crucial to investigate the effect of several parameters on

inactivation of pathogenic microorganisms in a real food matrix. Therefore, the efficacy

of pulsed UV-light on inactivation of S. aureus in milk was investigated.

The effects of sample volume, treatment time, and distance of the milk sample

from the quartz window were investigated for inactivation of S. aureus in milk.

Temperature of the milk was increased gradually during a pulsed UV-light treatment,

resulting in temperatures up to 91oC after a 3 min treatment. This clearly indicated that

there might be a synergistic effect of temperature increase on microbial inactivation.

Complete inactivation of S. aureus was achieved when 30 mL of inoculated milk was

treated for 180 s at 8 cm distance from the quartz window and when 12 mL of inoculated

milk was treated for 180 s at 10.5 cm distance from the quartz window. The

corresponding reduction was 8.55 log10 CFU/ml. A surface response model predicted the

log10 reduction reasonably with an R2 of 0.87. The model can be further improved, if the

experimental design can incorporate the effect of other variables such as blocking of UV-

light by food components, temperature increases etc. Results clearly demonstrated that

pulsed UV-light can be utilized for milk treatment. However, to test the feasibility of

pulsed UV-light for industrial milk treatment application, one has to investigate the

efficacy of pulsed UV-light for continuous treatment of milk. Furthermore, milk quality

169

may change because of pulsed UV-light treatments have to be monitored to ensure there

is no adverse quality effect. To investigate this possibility, milk inoculated with S.

aureus was pumped using a peristaltic pump and treated with pulsed UV-light.

S. aureus was artificially inoculated to contaminate raw milk, which was then and

pumped through a quartz tube placed in the pulsed UV-light chamber and treated. The

effects of distance from the quartz window, number of passes, and flow rate were

investigated. Reductions obtained from the treatments varied from 0.55 to 7.26 log10

CFU/ml. Complete inactivation of S. aureus was obtained at 1) 8 cm sample distance

from the quartz window, single pass, and 20 ml/min flow rate, and 2) 11 cm sample

distance from the quartz window, 2 passes, and 20 ml/min flow rate combinations. The

reduction corresponding to complete inactivation was more than 7 log10 CFU/ml. The

temperature of the milk sample rose up to 43oC during the treatment indicating that there

might be a synergistic effect of temperature increase. A 29 panelists consumer panel

evaluated the pulsed UV-light treated skim and 1% milk samples. There was some

perceivable change in the milk flavor. A 9-point hedonic scale with 1 being ‘dislike

extremely’ and 9 being ‘like extremely’ was used. Generally, the pulsed UV-light treated

sample received 3 to 4 points less than the untreated pasteurized milk samples. The

pulsed UV-light treated whole milk had a mushroom like or burnt flavor, and thus was

not used in the evaluations. These flavor changes occurred mainly because of prolonged

pulsed UV-light exposure for treating a thick layer of milk.

Treating the milk as a thin film will ensure shorter treatment duration, which in

turn will reduce the quality changes. Pulsed UV-light can also be used in conjunction

170

with a thermal pasteurization system since it can inactivate pathogens which are resistant

to thermal treatment.

These studies clearly indicated that pulsed UV-light was effective in inactivating

vegetative cells of pathogenic microorganisms in agar seeded cells, phosphate buffer, and

milk. Treatments which inactivate vegetative cells might not inactivate spores of

pathogenic microorganisms. Spores are normally more resistant than vegetative cells and

hence very difficult to inactivate by current pasteurization methods. Therefore, the

efficacy of pulsed UV-light on inactivation of B. subtilis spores in water was

investigated.

An annular pulsed UV-light chamber with the lamp at the center was used in this

experiment. This design ensured that all the pulsed UV-light energy was utilized for

inactivation of bacterial spores because the energy was absorbed from 360o and reflected

back by the polished surface. To test the feasibility of the pulsed UV-light process for

industrial scale, higher flow rates were tested (up to 14 L/min). Complete inactivation of

B. subtilis spores was obtained at all tested flow rates up to 14 LPM resulting in

reductions up to 6.5 log10 CFU/ml. Enrichment in no-light and light proved that there

was no growth indicating that the spores were not able to recover by photorepair

mechanisms. Therefore, pulsed UV-light completely inactivated the bacterial spores by

not providing a chance to recover. Hence, pulsed UV-light has a potential to be used for

inactivation of bacterial spores. As B. subtilis is a surrogate of pathogenic strains such as

B. anthracis and B. cereus, these results suggest that pulsed UV-light potentially can

inactivate these pathogenic strains also.

171

The efficacy of infrared heating on inactivation of S. aureus was determined as a

comparison. Infrared heating has several advantages such as low energy costs, faster

heating rate, etc. Reductions of 0.10 to 8.41 log10 CFU/ml were obtained under the tested

conditions. Complete inactivation of S. aurues was achieved at two conditions. Further

enrichment indicated that some injured cells repaired themselves. Consequently, infrared

heating can likely be used to replace conventional heating systems, resulting in cost

reductions of the processing. Another advantage of infrared heating is that one can

selectively heat the target material, such as microorganism in the food material without

heating other food components. This achievement will ensure the quality of the food

material by not inducing any changes because of heat treatments.

When compared to pulsed UV-light treatment, infrared heating could be readily

applicable for the treatment of milk at the first glance since quality changes in pulsed

UV-light treatment were significant. However, pulsed UV-light has a potential to be

used for milk pasteurization provided detrimental wavelengths are filtered to maintain the

quality of milk. A further design of infrared heating chamber for continuous milk

treatment will result in better quality milk as it reduces the burnt flavor induced mainly

due to static milk treatment for a prolonged time at an elevated temperature.

As the flavor changes induced by infrared heat treatment might possibly similar to

the changes due to thermal pasteurization, infrared heat treatment might be easily

accepted by the consumers. However, light induced flavor changes during pulsed UV-

light treatment could cause unacceptability in consumers as indicated by the sensory

studies (Appendix B). Nevertheless, it is possible to filter out the wavelengths inducing

172

light induced flavors by proper treatment chamber and UV-lamp design which could

result in acceptable milk quality.

To optimize a disinfection process, it is crucial to understand the pathogen

inactivation mechanisms of novel technologies such as pulsed UV-light and infrared

heating. Therefore, the inactivation mechanism of pulsed UV-light and infrared heating

was investigated by Transmission Electron Microscopy (TEM) and Fourier transform

spectroscopy. The pulsed UV-light results showed that the inactivation mechanism is

different from continuous UV-light treatment, where inactivation is mainly because of

thymine dimer in the DNA. However, a 5-second treatment with pulsed UV-light

treatment resulted in cell wall breakage, cytoplasm leakage, damage in the cellular

membrane structure, and leakage of the cell content. The damage in the structure of the

cell might be caused because of the pulsing effect of pulsed UV-light since many of the

damage characteristics were analogous to pulsed electric field damage. Damages caused

by pulsed UV-light can be grouped into 1) photochemical damage: Thymine dimer

formation and other photochemical changes in DNA; 2) photothermal damage: Damage

caused to bacterial cell because of the differences in the heating rates of bacteria and the

surrounding media resulting in localized heating of bacterial cell; 3) pulsed effect

damage: Damage because of disturbances caused by intermittent pulses. In case of

infrared heating, condensation of cytoplasm, shrinkage of cytoplasmic membrane, cell

wall damage, and cellular content leakage occurred after an infrared heating treatment.

The pulsed UV-light and infrared heat treated samples were successfully classified by

FTIR micro-spectrometry and discriminate analysis. Absorption bands in the infrared

region were also successfully assigned to the treated cells.

173

Results of these studies are limited to the conditions tested in the study. As novel

technologies, pulsed UV-light and infrared heating are still in developing stage s; hence

several improvements can be made. For pulsed UV-light treatment, monitoring of the

actual energy absorbed by the food sample at different depth levels will be beneficial for

model development and process validation. During prolonged pulsed UV-light treatment,

temperature of the sample increased. The effects of thermal inactivation should be

monitored and recognized. For certain products, temperature build-up can be detrimental

to quality. Hence, filtering out the wavelengths causing temperature increases could help

preserve the quality of the food. As a broadband spectrum light, pulsed UV-light also

includes the wavelength range from 330 to 480 nm which are responsible for

photoreactivation, a mechanism to repair the DNA damage caused by UV-light. It will

be crucial to filter out this wavelength region in order to ensure the complete inactivation

of pathogenic microorganisms. For continuous treatment of liquid food by pulsed UV-

light, a mechanism has to be developed to treat a food material as a thin film for reducing

treatment time and enhancing quality. For opaque food materials, treatment with some

photocatalyzer may enhance effectiveness of pulsed UV-light treatment. Provision of a

heat sink such as cold air can be beneficial to preserve the quality of the food material by

avoiding quality changes caused by temperature build-up. The annular pulsed UV-light

system can be modified for treating clear food materials such as apple juice by reducing

the annular space. Extensive studies on pathogen inactivation have to be continued in

order to identify the most resistant pathogen. An easier and faster way to determine the

adequacy of the pulsed UV-light treatments by measuring absorbed UV-light energy has

to be developed.

174

A user friendly ‘Graphical User Interface (GUI)’ with an option for precise

control would be beneficial for controlling the infrared lamps. To cool the filter for

selective heating, an integrated fan control should be included. Since there are very

limited studies on microbial inactivation by infrared heating, the effect of infrared heating

on inactivation of pathogenic microorganisms has to be investigated. Extensive sensory

evaluation studies have to be done to investigate the possible off-flavors produced during

infrared heating and effective elimination of those off-flavors. A predictive model for

microbial inactivation has to be developed. Effect of parameters such as composition of

food (level of fat content, moisture content, etc), number of infrared lamps, orientation of

lamps, type of lamps, and distance of sample from the lamps has to be investigated.

Further detailed study on inactivation mechanism can be tested for pulsed UV-

light and infrared heating. Furthermore, the efficacy of the pulsed UV-light can be

compared to equivalent energy continuous UV-light and infrared heating to conventional

heating in order to investigate potential benefits of these techniques. Detailed study of

inactivation of other pathogenic microorganisms and spores by these techniques for

different food products could be helpful in designing sterilization equipment.

Development of precise prediction models would be helpful for better estimation of

processing parameters.

175

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Appendix A. Inactivation Kinetics of Pulsed UV-light

Treatment of Staphylococcus aureus

UV dose-response curves for dispersed or free-floating microorganisms can be

represented by first-order kinetics (EPA, 2003; Severin et al., 1984; Liu, 2005).

Generally, an UV-light inactivation curve is sigmoidal (CFSAN-FDA, 2000) with a

shoulder and tail. Shoulder effect is due to delayed response of a microorganism to UV-

light because of injury (EPA, 2003; CFSAN-FDA, 2000). Photo-reactivation, dark

repair, resistance of bacteria are factors influencing shoulder effect. Tailing effect occurs

because of shielding of external particles, clumping of bacteria, and resistant

microorganisms. Pulsed UV-light has very high instantaneous energy as compared to

continuous UV-light. Even a second treatment results in significant inactivation of

pathogenic microorganism. Several researchers suggested that inactivation of pathogens

occur within seconds because of increased energy availability. Therefore, pulsed UV-

light inactivation curves may not have a shoulder because of high instantaneous energy

available for microbial inactivation. Pulsed UV-light may also not have tail effect for

inactivation of pathogens in a clear solution. However, while treating a complex food

matrix with particles, one may need to take into account these effects. Inactivation

models developed for UV-light can be utilized for pulsed UV-light modeling since a

significant portion of the energy delivered in a pulsed UV-light system is in the UV range

(56% of the total energy was in UV range for the germicidal pulsed UV-light lamp used

in this study).

185

The first order inactivation equation (Severin et al., 1984) can be represented as

)**(exp tIkoNN −= (Equation 1)

where,

N = Concentration of viable microorganisms after UV-light treatment (CFU/ml)

No = Concentration of viable microorganisms before UV-light treatment

(CFU/ml)

k = First order inactivation coefficient (cm2/J)

I = Intensity of UV-light energy applied (J/cm2)

t = Treatment time (s)

Equation 1 can also be represented as (EPA, 2003)

⎟⎟⎠

⎞⎜⎜⎝

⎛−

− == 1010exp )( DD

kDoNN (Equation 2)

where,

D = I*t = UV dose delivered or fluence rate (J/cm2).

D10 = UV dose required to achieve 90% reduction in microbial population.

Therefore,

10

logDD

NNo =⎟

⎠⎞

⎜⎝⎛

(Equation 3)

It is important to know the D10 value of a microorganism to evaluate the extent of

the resistance of the microorganism to UV-light. Higher D10 values indicate that the

microorganism is very resistant to UV-light and require more energy for inactivation.

186

From equation 2,

)(exp kD

oNN −=

Therefore,

DkkDNNo *

303.2)10ln(1*log ⎟

⎠⎞

⎜⎝⎛==⎟

⎠⎞

⎜⎝⎛

(Equation 4)

⎟⎠⎞

⎜⎝⎛

NNolog ⎟

⎠⎞

⎜⎝⎛

303.2kWhere, is the log10 reduction of microbial population and is

the slope of the fitted straight line for the plot of log10 reduction vs. available UV dosage.

The pulsed UV-light doses received and the corresponding log10 reductions of S.

aureus in 0.1M phosphate buffer and agar seeded cell treatment were presented in Figure

1 (chapter 3). The samples were treated with pulsed UV-light for up to 30 s at 8 cm

distance from the quartz window (The centre axis of the lamp was located 5.8 cm above

the quartz window). However, only data from the first five seconds of treatment were

used for model development since complete inactivation was obtained after a 5-s

treatment (Figure A1). The broadband energy intensity available at this location was

measured to be 0.33 J/cm2/pulse using a radiometer (Ophir PE50, Ophir Optronics Inc.,

Wilmington, MA). In the case of phosphate buffer treatment, a 48 ml sample was treated

with pulsed UV-light.

187

y = 0.93x + 3.46R2 = 0.90

y = 0.27x2 - 0.65x + 5.29R2 = 1.00

y = 0.27x2 - 0.16x + 1.50R2 = 1.00

y = 1.47x - 0.37R2 = 0.95

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

0 1 2 3 4 5

UV dose (J/cm2)

Log 1

0 red

uctio

n (lo

g 10 C

FU/m

l)

6

Agar seeded cells

Phosphate buffer treatment

____ Linear fit line- - - - Quardratic fit line

Figure A1. Inactivation of S. aureus by pulsed UV-light treatment.

The log10 reduction was plotted against estimated irradiation dosage available to

the microorganism and a linear and quadratic equation was fitted. The D10 values and

rate constants (first order inactivation coefficient, k) were estimated using a linear fit with

Equations 2 and 4, respectively (Table A1).

Table A1. Estimation of inactivation model parameters.

Treatment D10 value (J/cm2) k (cm2/J) R2

Phosphate buffer treatment 0.68 2.15 0.95 Agar seed cells 1.07 3.38 0.90

The D10 value of the agar seeded cells was higher because of ~5 log10 reduction

obtained within 1 s, resulting in reduction in the slope of the equation. The reduction in

microbial population after 1 s treatment was slower for additional UV dose supplied as

188

compared to the phosphate buffer treatment. The first order inactivation coefficient (k)

values for S. aureus treated in phosphate buffer and as agar seeded cells were 3.38, and

2.15 cm2/J, respectively (Table A1). The reduction was gradual in case of phosphate

buffer treatment. Though the linear curve fitting explained more than 90% of the

variation in data, quadratic curve fitting resulted in R2 of approximately 1.00, indicating

that dose response of pulsed UV-light was quadratic (Table A1).

When the microorganisms exhibit a shoulder effect, Equation 2 can be modified

as , where, d is the intercept of the exponential phase

of the dose-response curve with the y-axis (EPA, 2003). Similarly, equation 2 can be

modified to take into account the tailing effect as follows: ,

where, No is the concentration of dispersed microorganisms present, Np is the

concentration of particles containing microorganisms, and kp is the inactivation constant

for microorganisms associated with particles (EPA, 2003). In this study, the shoulder and

tail effects are not applicable as there was no indication of either a shoulder or a tail in the

dose-response curve. Because of availability of sufficient energy, there was no shoulder

effect as indicated by 4.91 and 1.55 log10 CFU/ml reduction of S. aureus within an 1-

second treatment. Furthermore, there was no growth observed after a 5-s treatment,

indicating that there was no resistant microbial population surviving. Even following the

enrichment procedure, no growth was observed, suggesting that there were no injured

cells.

dkDoNN )exp1(1( )(−−−=

)()( Dkp

kDo

peNeNN −− +=

189

References

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190

http://www.cfsan.fda.gov/%7Ecomm/ift-uv.html

Appendix B. Sensory Evaluation of Pulsed UV-light

Treated Milk

It is crucial to monitor the changes in sensory attributes of milk during pulsed

UV-light treatment to ensure the commercial applicability. Therefore, the milk treated

with pulsed UV-light was evaluated by consumer panelists. The panelists rated the

overall liking on a 9-point hedonic scale.

Materials and methods

Pasteurized milk was used instead of raw milk for pulsed UV-light treatments in

order to ensure the safety of the consumers. Therefore, pasteurized milk was further

treated with pulsed UV-light to test the effect of UV-light on perceivable changes in the

overall acceptability of the product. In order to demonstrate the effect of fat content,

skim milk and 1% milk were used. Both treated and untreated samples were stored in the

refrigerator (4oC) overnight. In order to determine overall acceptability on a 9-point

hedonic scale (9 being like extremely and 1 being dislike extremely), about 15 ml of each

refrigerated samples were served in sample cups with 3-digit blinding code and served to

the panelist in a randomized serving order generated by the Compusense software

(Compusense®; Ontario, Canada). Panelists were selected based on the criteria that they

are consumers of milk.

A 9-point hedonic scale (1 being ‘dislike extremely’ and 9 being ‘like extremely’)

was used to evaluate the overall acceptability of the milk products before and after pulsed

UV-light treatment. A consumer panel of 29 panelists evaluated the products. Panelists

191

received the samples with 3-digit blind codes in a randomized serving order to reduce

biases. Compusense® software was used to design and analyze the consumer panel test.

The results clearly show that the UV-light treated milk induce some perceivable

change in the flavor of the milk and thus makes the product comparatively less

acceptable. Preliminary sensory evaluation studies indicated that the pulsed UV-light

treated whole milk had a distinct burnt flavor and/or mushroom soup flavor based on the

input from trained sensory panelists. Therefore, only 1% and skim milk were used for

further sensory studies. In general, the pulsed UV-light treated milk samples were rated

3 to 4 points less than the untreated samples (Table A2).

Table A2. Sensory evaluation of UV treated milk (n=29).

Overall acceptability1,2 Skim milk 1% fat milk Control (pasteurized) 5.76 ± 1.83A 6.24 ± 2.10A UV-light treated (pasteurization followed by UV-light treatment)

1.79 ± 1.29B 2.10 ± 1.52B

1A 9-point hedonic scale was used, where 1 = dislike extremely, and 9 = like extremely. 2Mean of 29 observations are given with the standard deviation. 3Means followed by different letter are significantly different from each other in the same column (p ≤ 0.05).

The variation between the samples and judges were statistically significant

(p<0.05). However, it is interesting to note that the majority of the consumers rated the

control (pasteurized milk) as mostly ‘neither like nor dislike’ or ‘like slightly’. This

clearly indicates that there are some flavor changes associated with the pasteurization

process which influenced the acceptability rating for the UV-light treated milk samples as

the pasteurized milk was further processed with pulsed UV-light. Flavor changes

induced by pasteurization also contribute to the lower acceptability rating. The ratings

192

for pulsed UV-light treated skim milk and 1% milk were 1.79 ± 1.29 and 2.10 ± 1.52,

respectively, while the control samples had a rating of 5.76 ± 1.83 and 6.24 ± 2.10,

respectively.

Previous research shows that pulsed UV-light can inactivate pathogens in a very

short time (up to several seconds exposure) if the thickness of the food product is

minimal. A setup which will allow milk to flow as a thin (1-3 mm thickness) film will be

helpful in reducing the exposure time as the penetration efficiency increases

exponentially, which will in turn reduce the treatment time, which will also reduce the

changes in the sensory quality of the milk.

In conclusion, sensory evaluation of pulsed UV-light treated and untreated milk

was performed. The treated milk received slightly lower rating than the untreated milk

indicating that there was some change to the sensory attributes. However, by optimizing

the process parameters, one can further improve the quality of milk.

193

Appendix C. Source Code: Infrared Heating Control Logic

#include <utility.h> #include <ansi_c.h> #include <formatio.h> #include <rs232.h> #include <cvirte.h> /* Needed if linking in external compiler; harmless otherwise */ #include <userint.h> #include "6lamps.h" #define OFF 0 #define ON 1 #define LPT1 0x378 char word; char set[10]; double sum=0; double save=0; int counter=0; int sign; int end=0; int dim1=1,dim2=1,dim3=1;dim4=1;dim5=1;dim6=1; double save1=0; //char TEMPSTR[7]; char buf[260]; int index_num; char sendword[30]; static int panelHandle; static int configHandle; static int choiceHandle; static int setHandle; static int graphHandle; char proj_dir[256]; char file_name[300]; static int go_num1,go_num2,go_num3,go_num4,go_num5,go_num6; int read; int choice1; int choice2; int loop1=0; int loop2=0; int loop3=0; int fan; int onandoff1, onandoff2, onandoff3, onandoff4,onandoff5, onandoff6; double readings1,readings2,readings3,readings4,readings5,readings6; int comport = 1, baudrate = 9600, parity = 0,

194

databits = 8, stopbits = 1; char Rsbuf[1000]; int datanum=0; int goflag=0; int goflag1=0; int goflag2=0; int temp; int stringsize,bytes_sent; double datapoints[15][20000]; double graphdata1[3],graphdata2[3],graphdata3[3],graphdata4[3] ,graphdata5[4],graphdata6[2],graphdata7[6]; typedef struct { int channel; double data[15]; int unit; } DataType; DataType Get; double calib(double x); void send_data(void); void receive_data(void); void SetConfigParms1 (void); void GetConfigParms1 (void); void Control_GK(void); void Control_New(void); void Control_New1(void); int ComRdStr(int port, char *s, int term) { int temp, ret = 0; while ((temp = ComRdByte(port)) != term) { if (temp == -99) break; s[ret++] = (char)temp; } s[ret] = '\0'; return ret; } int main (int argc, char *argv[]) { if (InitCVIRTE (0, argv, 0) == 0) /* Needed if linking in external compiler; harmless otherwise */ return -1; /* out of memory */ if ((panelHandle = LoadPanel (0, "6lamps0228.uir", PANEL)) < 0) return -1; graphHandle = LoadPanel (0, "6lamps0228.uir",GRAPH); DisplayPanel (panelHandle);

195

SetCtrlAttribute(panelHandle,PANEL_STOPBUTTON,ATTR_VISIBLE,0); RunUserInterface (); return 0; } int CVICALLBACK ProceedCall (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { int i,ret=0; char Temp[10]; switch (event) { case EVENT_COMMIT: SetCtrlAttribute(panelHandle,PANEL_STOPBUTTON,ATTR_VISIBLE,1); SetCtrlAttribute(panelHandle,PANEL_GOBUTTON,ATTR_VISIBLE,0); DisplayPanel (graphHandle); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6_1,ATTR_VISIBLE,0); OpenComConfig (comport, "", baudrate, parity, databits, stopbits, 1024, 512); /* strcpy(sendword, "INIT\n"); stringsize = StringLength(sendword); bytes_sent = ComWrt (comport, sendword, stringsize); strcpy(sendword, "FETCh?\n"); stringsize = StringLength(sendword); bytes_sent = ComWrt (comport, sendword, stringsize); */ goflag =1; break; } return 0; } void send_data(void) { int j; int ratio=0; /* strcpy(sendword, "READ?\n"); stringsize = StringLength(sendword); bytes_sent = ComWrt (comport, sendword, stringsize); */ strcpy(sendword, "INIT\n"); stringsize = StringLength(sendword); bytes_sent = ComWrt (comport, sendword, stringsize); strcpy(sendword, "FETCh?\n"); stringsize = StringLength(sendword); bytes_sent = ComWrt (comport, sendword, stringsize);

196

ComRdStr(comport, Rsbuf, 10); } void receive_data(void) { int j=0; int k=0; int l=0; int ratio=0; //ComRd (comport, Rsbuf, 200); if (choice1) { Scan(Rsbuf, "%s[i14w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i1w6]>%f[p4]", &(Get.data[0])); Get.data[0]= Get.data[0]*ratio; Scan(Rsbuf, "%s[i30w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i17w6]>%f[p4]", &(Get.data[1])); Get.data[1]= Get.data[1]*ratio; Scan(Rsbuf, "%s[i46w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i33w6]>%f[p4]", &(Get.data[2])); Get.data[2]= Get.data[2]*ratio; Scan(Rsbuf, "%s[i62w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i49w6]>%f[p4]", &(Get.data[3])); Get.data[3]= Get.data[3]*ratio; Scan(Rsbuf, "%s[i110w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i97w6]>%f[p4]", &(Get.data[4])); Get.data[4]= Get.data[4]*ratio; Scan(Rsbuf, "%s[i174w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i161w6]>%f[p4]", &(Get.data[5])); Get.data[5]= Get.data[5]*ratio; Scan(Rsbuf, "%s[i190w1]>%i", &(Get.unit)); if (Get.unit==1) ratio=10; if (Get.unit==2) ratio=100; Scan(Rsbuf, "%s[i177w6]>%f[p4]", &(Get.data[6])); Get.data[6]= Get.data[6]*ratio;

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} if (choice2) { Scan(Rsbuf, "%s>%15f[x]", Get.data); } } int CVICALLBACK CloseMainCall (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: SetCtrlVal (panelHandle, PANEL_LED, OFF); CloseCom (comport); outp(LPT1,0x00); QuitUserInterface (0); go_num1 =0; go_num2 =0; go_num3 =0; go_num4 =0; break; } return 0; } int CVICALLBACK timer_control (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { int i; switch (event) { case EVENT_TIMER_TICK: if (goflag ==1) { // SetSystemAttribute (ATTR_ALLOW_UNSAFE_TIMER_EVENTS, 1); SetCtrlVal (panelHandle, PANEL_LED, ON); send_data(); SetCtrlVal (panelHandle, PANEL_LED, OFF); goflag1=1; } break; } return 0; } int CVICALLBACK timer2_control (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) {

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case EVENT_TIMER_TICK: // SetSystemAttribute (ATTR_ALLOW_UNSAFE_TIMER_EVENTS, 1); if (goflag1 ==1) { receive_data(); goflag2 =1; Control_New(); } break; } return 0; } int CVICALLBACK end_job (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT:

SetCtrlAttribute(panelHandle,PANEL_STOPBUTTON,ATTR_VISIBLE,0); SetCtrlAttribute(panelHandle,PANEL_GOBUTTON,ATTR_VISIBLE,1); SetCtrlVal (panelHandle, PANEL_LED, OFF); SetCtrlVal (panelHandle, PANEL_LED1, OFF); SetCtrlVal (panelHandle, PANEL_LED2, OFF); SetCtrlVal (panelHandle, PANEL_LED3, OFF); SetCtrlVal (panelHandle, PANEL_LED4, OFF); SetCtrlVal (panelHandle, PANEL_LED5, OFF); SetCtrlVal (panelHandle, PANEL_LED6, OFF);

CloseCom (comport); SetCtrlVal (panelHandle, PANEL_FAN, OFF);

outp(LPT1,0x00); goflag = 0; goflag1=0; goflag2=0; go_num1 =0; go_num2 =0; go_num3 =0; go_num4 =0; sum=0; save=0; end =1; break; } return 0; } int Clear (int panel, int control, int event, void *callbackData, int eventData1, int eventData2)

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{ int i,j, readings; switch (event) { case EVENT_COMMIT: /* DeleteListItem (panelHandle, PANEL_TEMP_LIST, 0, 0);*/ ClearListCtrl (panelHandle, PANEL_TEMP_LIST); datanum=0; ClearStripChart (graphHandle, GRAPH_TEMP_GRAPH1); ClearStripChart (graphHandle, GRAPH_TEMP_GRAPH2); ClearStripChart (graphHandle, GRAPH_TEMP_GRAPH3); ClearStripChart (graphHandle, GRAPH_TEMP_GRAPH4); ClearStripChart (graphHandle, GRAPH_TEMP_GRAPH5); ClearStripChart (graphHandle, GRAPH_TEMP_GRAPH6); ClearStripChart (panelHandle, PANEL_TEMP_GRAPH7); /* DeleteGraphPlot (panelHandle, PANEL_TEMP_GRAPH, -1, VAL_DELAYED_DRAW); */ SetCtrlVal (panelHandle, PANEL_LED1, OFF); SetCtrlVal (panelHandle, PANEL_LED2, OFF); SetCtrlVal (panelHandle, PANEL_LED3, OFF); SetCtrlVal (panelHandle, PANEL_LED4, OFF); SetCtrlVal (panelHandle, PANEL_LED5, OFF); SetCtrlVal (panelHandle, PANEL_LED6, OFF); // goflag=0; // goflag1=0; SetCtrlVal (panelHandle, PANEL_TEST2, " "); for( i =0;i<1000;i++) { j++; } SetCtrlVal (panelHandle, PANEL_CLEAR, OFF); SetCtrlVal (panelHandle, PANEL_LED, OFF); outp(LPT1,(0x00|word)); go_num1 =0; go_num2 =0; go_num3 =0; go_num4 =0; end =1; break; } return 0; } int Save(int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { FILE *filehandle; int i; switch ( event) {

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case EVENT_COMMIT: FileSelectPopup (proj_dir, "*.txt", "*.txt", "Save As", VAL_OK_BUTTON, 0, 1, 1, 0, file_name) ; filehandle = fopen (file_name, "w+"); fprintf(filehandle, "RS232C OPERATING SYSTEM \n\n"); fprintf(filehandle,"*********< Information >*********\n"); fprintf(filehandle, "Channel Acquired: LAMP1 LAMP2 LAMP3 LAMP4 LAMP5 LAMP6 SAMP1 SAMP2\n"); fprintf(filehandle, "Date %s Time: %s \n", DateStr(), TimeStr()); fprintf (filehandle, "----------------------------------\n"); for (i=0;i<datanum;i++) { fprintf (filehandle, "%d : %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f %4.1f\n", i,datapoints[0][i], datapoints[1][i],datapoints[2][i],datapoints[3][i],datapoints[4][i],datapoints[5][i],datapoints[6][i],datapoints[7][i],datapoints[8][i],datapoints[9][i],datapoints[10][i],datapoints[11][i],datapoints[12][i]); } fclose (filehandle); } return (0); } int CVICALLBACK ConfigCall (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: configHandle = LoadPanel (panelHandle, "6lamps0228.uir",CONFIG); // SetCtrlVal(configHandle, CONFIG_SETWHAT, "Setting Hydro Thermometer"); InstallPopup (configHandle); SetConfigParms1(); break; } return 0; } void SetConfigParms1 (void) { SetCtrlVal (configHandle, CONFIG_COMPORT, comport); SetCtrlVal (configHandle, CONFIG_BAUDRATE, baudrate); SetCtrlVal (configHandle, CONFIG_PARITY, parity); SetCtrlVal (configHandle, CONFIG_DATABITS, databits); SetCtrlVal (configHandle, CONFIG_STOPBITS, stopbits); }

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/********************************************************************/ void GetConfigParms1 (void) { GetCtrlVal (configHandle, CONFIG_COMPORT, &comport); GetCtrlVal (configHandle, CONFIG_BAUDRATE, &baudrate); GetCtrlVal (configHandle, CONFIG_PARITY, &parity); GetCtrlVal (configHandle, CONFIG_DATABITS, &databits); GetCtrlVal (configHandle, CONFIG_STOPBITS, &stopbits); } int CloseConfigCallback (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: choiceHandle = LoadPanel (panelHandle, "6lamps0228.uir",CHOICE); GetConfigParms1(); DiscardPanel (configHandle); InstallPopup (choiceHandle); break; } return 0; } int CVICALLBACK Closechoice (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: setHandle = LoadPanel (panelHandle, "6lamps0228.uir",SET); GetCtrlVal (choiceHandle, CHOICE_CHECKBOX1, &choice1); GetCtrlVal (choiceHandle, CHOICE_CHECKBOX2, &choice2); if (choice1 == choice2) { MessagePopup ("Error Message", "You must choose either of two systems"); }else { DiscardPanel (choiceHandle); if (choice1) { SetCtrlAttribute(setHandle,SET_SETPOINT_5,ATTR_VISIBLE,0); SetCtrlAttribute(setHandle,SET_BINARYSWITCH5,ATTR_VISIBLE,0); SetCtrlAttribute(setHandle,SET_RING5,ATTR_VISIBLE,0); SetCtrlAttribute(setHandle,SET_SETPOINT_6,ATTR_VISIBLE,0); SetCtrlAttribute(setHandle,SET_BINARYSWITCH6,ATTR_VISIBLE,0);

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SetCtrlAttribute(setHandle,SET_RING6,ATTR_VISIBLE,0); } InstallPopup (setHandle); } break; } return 0; } int CVICALLBACK CloseConfigCallback1 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { int i; double j; char set[20]; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT, &readings1); graphdata1[1] = readings1; GetCtrlVal (setHandle, SET_SETPOINT_2, &readings2); graphdata2[1] = readings2; GetCtrlVal (setHandle, SET_SETPOINT_3, &readings3); graphdata3[1] = readings3; GetCtrlVal (setHandle, SET_SETPOINT_4, &readings4); graphdata4[1] = readings4; GetCtrlVal (setHandle, SET_SETPOINT_5, &readings5); graphdata5[1] = readings5; GetCtrlVal (setHandle, SET_SETPOINT_6, &readings6); graphdata6[1] = readings6; GetCtrlVal (setHandle, SET_BINARYSWITCH1, &onandoff1); GetCtrlVal (setHandle, SET_BINARYSWITCH2, &onandoff2); GetCtrlVal (setHandle, SET_BINARYSWITCH3, &onandoff3); GetCtrlVal (setHandle, SET_BINARYSWITCH4, &onandoff4); GetCtrlVal (setHandle, SET_BINARYSWITCH5, &onandoff5); GetCtrlVal (setHandle, SET_BINARYSWITCH6, &onandoff6); DiscardPanel (setHandle); Fmt(set, "%s<%f[p2]", readings1); SetCtrlVal (panelHandle, PANEL_STRING1, set); Fmt(set, "%s<%f[p2]", readings2); SetCtrlVal (panelHandle, PANEL_STRING2, set); Fmt(set, "%s<%f[p2]", readings3); SetCtrlVal (panelHandle, PANEL_STRING3, set); Fmt(set, "%s<%f[p2]", readings4); SetCtrlVal (panelHandle, PANEL_STRING4, set); Fmt(set, "%s<%f[p2]", readings5); SetCtrlVal (panelHandle, PANEL_STRING5, set); Fmt(set, "%s<%f[p2]", readings6);

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SetCtrlVal (panelHandle, PANEL_STRING6, set); SetCtrlAttribute(panelHandle,PANEL_GOBUTTON,ATTR_DIMMED,0); SetCtrlAttribute(panelHandle,PANEL_CONFIG_CALL,ATTR_DIMMED,1); if (choice2) { SetCtrlVal (panelHandle, PANEL_TEST1, "New Heating System"); SetCtrlAttribute(panelHandle,PANEL_FAN,ATTR_DIMMED,0); }

if (choice1) SetCtrlVal (panelHandle, PANEL_TEST1, "Old Heating System");

if (onandoff1 ==0) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1,ATTR_DIMMED,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1_1,ATTR_DIMMED,1); dim1=0; } if (onandoff2 ==0) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2,ATTR_DIMMED,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2_1,ATTR_DIMMED,1); dim2=0; } if (onandoff3 ==0) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3,ATTR_DIMMED,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3_1,ATTR_DIMMED,1); dim3=0; } if (onandoff4 ==0) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4,ATTR_DIMMED,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4_1,ATTR_DIMMED,1); dim4=0; } if (onandoff5 ==0) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5,ATTR_DIMMED,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5_1,ATTR_DIMMED,1); dim5=0; } if (onandoff6 ==0) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6,ATTR_DIMMED,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6_1,ATTR_DIMMED,1); dim6=0; }

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break; } return 0; } double calib(double x) { return (2898/(x+273)); } double decalib(double x) { return (2898/x -273); } int CVICALLBACK setpoint1 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { char TEMPSTR[7]; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT, &readings1); SetCtrlVal(setHandle, SET_RING1, calib(readings1)); break; } return 0; } int CVICALLBACK setpoint2 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { char TEMPSTR[7]; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT_2, &readings2); SetCtrlVal(setHandle, SET_RING2, calib(readings2)); break; } return 0; } int CVICALLBACK setpoint3 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { char TEMPSTR[7]; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT_3, &readings3); SetCtrlVal(setHandle, SET_RING3, calib(readings3)); break; }

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return 0; } int CVICALLBACK setpoint4 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT_4, &readings4); SetCtrlVal(setHandle, SET_RING4, calib(readings4)); break; } return 0; } int CVICALLBACK setpoint5 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT_5, &readings5); SetCtrlVal(setHandle, SET_RING5, calib(readings5)); break; } return 0; } int CVICALLBACK setpoint6 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_SETPOINT_6, &readings6); SetCtrlVal(setHandle, SET_RING6, calib(readings6)); break; } return 0; } void Control_New(void) { int on_num1,on_num2,on_num3,on_num4,on_num5,on_num6; // char TEMPSTR[7]; double dty1,dty2,dty3,dty4,dty5,dty6,dy1,dy2,dy3,dy4,dy5,dy6; char word1,word2, word3, word4, word5, word6,word7; char sent_word1[30]; char sent_word; dy1 = readings1 - Get.data[0]; /*dy1 is the differnce between set temperature (readings1) and the actual temperature (Get.data[0])- Kadir */

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dy2 = readings2 - Get.data[1]; dy3 = readings3 - Get.data[2]; dy4 = readings4 - Get.data[3]; dy5 = readings5 - Get.data[4]; dy6 = readings6 - Get.data[5]; dty1 = ((datanum > 1) ? datapoints[0][datanum - 1] - datapoints[0][datanum - 2] : 0.0); /* dty1 tries to check there is a difference between the temperature collected in the previous time step and the current time step. If there is a difference exist it will adjust the on_num value to compensate for that - Kadir*/ dty2 = ((datanum > 1) ? datapoints[1][datanum - 1] - datapoints[1][datanum - 2] : 0.0); dty3 = ((datanum > 1) ? datapoints[2][datanum - 1] - datapoints[2][datanum - 2] : 0.0); dty4 = ((datanum > 1) ? datapoints[3][datanum - 1] - datapoints[3][datanum - 2] : 0.0); dty5 = ((datanum > 1) ? datapoints[4][datanum - 1] - datapoints[4][datanum - 2] : 0.0); dty6 = ((datanum > 1) ? datapoints[5][datanum - 1] - datapoints[5][datanum - 2] : 0.0); //number 1 if(readings1 > 500) { if (dy1 < 0 ) on_num1 = 0; else if (Get.data[0] > (0.999*readings1)) on_num1 = 1; else if (Get.data[0] > (0.997*readings1)) on_num1 = 3; /* else if (Get.data[0] > (0.997*readings1)) on_num1 = 5; // else if (Get.data[0] > (0.996*readings1)) on_num1 = 7; // else if (Get.data[0] > (0.994*readings1)) on_num1 = 9; // else if (Get.data[0] > (0.992*readings1)) on_num1 = 10; else if (Get.data[0] > (0.99*readings1)) on_num1 = 11;*/ else if (Get.data[0] > (0.95*readings1)) on_num1 = 10; /* else if (Get.data[0] > (0.9*readings1)) on_num1 = 14; else if (Get.data[0] > (0.8*readings1)) on_num1 = 15; */ else on_num1 = 16; if((dy1 < 10.0) && (dy1 > -5)) //Kadir changed it to 10 (initially it was 80 (dy1) { if (dty1 < -1.0) on_num1 += 16; else if (dty1 < -0.7) on_num1 += 12; else if (dty1 < -0.2) on_num1 += 8; } }

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else if(readings1 > 300) { if (dy1 < 0 ) on_num1 = 0; else if (Get.data[0] > (0.995*readings1)) on_num1 = 2; else if (Get.data[0] > (0.99*readings1)) on_num1 = 3; else if (Get.data[0] > (0.96*readings1)) on_num1 = 3; else if (Get.data[0] > (0.9*readings1)) on_num1 = 4; else if (Get.data[0] > (0.85*readings1)) on_num1 = 4; else if (Get.data[0] > (0.8*readings1)) on_num1 = 5; else if (Get.data[0] > (0.7*readings1)) on_num1 = 6; else if (Get.data[0] > (0.6*readings1)) on_num1 = 7; else on_num1 = 8; if((dy1 < 80.0) && (dy1 > -10)) { if (dty1 < -2.0) on_num1 += 6; else if (dty1 < -0.7) on_num1 += 4; else if (dty1 < -0.2) on_num1 += 3; else if (dty1 < 0) on_num1 += 2; } } else if ( readings1 > 150) { if (dy1 < 0 ) on_num1 = 0; else if (Get.data[0] > (0.99*readings1)) on_num1 = 0; else if (Get.data[0] > (0.96*readings1)) on_num1 = 2; else if (Get.data[0] > (0.9*readings1)) on_num1 = 3; else if (Get.data[0] > (0.85*readings1)) on_num1 = 4; else if (Get.data[0] > (0.8*readings1)) on_num1 = 5; else if (Get.data[0] > (0.7*readings1)) on_num1 = 6; else if (Get.data[0] > (0.6*readings1)) on_num1 = 7; else on_num1 = 8; if((dy1 > -1)&& (dty1 < 0)) on_num1 += 2; } else { if (dy1 < 0 ) on_num1 = 0; else if (Get.data[0] > (0.99*readings1)) on_num1 = 0; else if (Get.data[0] > (0.96*readings1)) on_num1 = 0; else if (Get.data[0] > (0.9*readings1)) on_num1 = 1; else if (Get.data[0] > (0.85*readings1)) on_num1 = 2; else if (Get.data[0] > (0.8*readings1)) on_num1 = 3; else if (Get.data[0] > (0.7*readings1)) on_num1 = 4;

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else if (Get.data[0] > (0.6*readings1)) on_num1 = 5; else on_num1 = 7; if((dy1 > -1)&& (dty1 < 0)) on_num1 += 2; } //on_num1 = 16; go_num1++; go_num1 = go_num1 % 16; if ((go_num1 < on_num1) &&(onandoff1)) // if (onandoff1) { word1=0x01; SetCtrlVal (panelHandle, PANEL_LED1, ON); } else { word1=0x00; SetCtrlVal (panelHandle, PANEL_LED1, OFF); } // number 2 if(readings2 > 500) { if (dy2 < 0 ) on_num2 = 0; else if (Get.data[1] > (0.999*readings2)) on_num2 = 1; else if (Get.data[1] > (0.998*readings2)) on_num2 = 3; /* else if (Get.data[1] > (0.997*readings2)) on_num2 = 5; // else if (Get.data[1] > (0.996*readings2)) on_num2 = 7; // else if (Get.data[1] > (0.994*readings2)) on_num2 = 9; // else if (Get.data[1] > (0.992*readings2)) on_num2 = 10; else if (Get.data[1] > (0.99*readings2)) on_num2 = 13;*/ else if (Get.data[1] > (0.95*readings2)) on_num2 = 10; /* else if (Get.data[1] > (0.9*readings2)) on_num2 = 15; else if (Get.data[1] > (0.75*readings2)) on_num2 = 15; */ else on_num2 = 16; if((dy2 < 10.0) && (dy2 > -5)) //Kadir - Changed dy2<80.0 to dy2<10.0 { if (dty2 < -1.0) on_num2 += 16; else if (dty2 < -0.7) on_num2 += 12; else if (dty2 < -0.2) on_num2 += 8; } } else if(readings2 > 300) { if (dy2 < 0 ) on_num2 = 0;

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else if (Get.data[1] > (0.995*readings2)) on_num2 = 2; else if (Get.data[1] > (0.99*readings2)) on_num2 = 3; else if (Get.data[1] > (0.96*readings2)) on_num2 = 3; else if (Get.data[1] > (0.9*readings2)) on_num2 = 4; else if (Get.data[1] > (0.85*readings2)) on_num2 = 4; else if (Get.data[1] > (0.8*readings2)) on_num2 = 5; else if (Get.data[1] > (0.7*readings2)) on_num2 = 6; else if (Get.data[1] > (0.6*readings2)) on_num2 = 7; else on_num2 = 8; if((dy2 < 80.0) && (dy2 > -10)) { if (dty2 < -2.0) on_num2 += 6; else if (dty2 < -0.7) on_num2 += 4; else if (dty2 < -0.2) on_num2 += 3; else if (dty2 < 0) on_num2 += 2; } } else if ( readings2 > 150) { if (dy2 < 0 ) on_num2 = 0; else if (Get.data[1] > (0.99*readings2)) on_num2 = 0; else if (Get.data[1] > (0.96*readings2)) on_num2 = 2; else if (Get.data[1] > (0.9*readings2)) on_num2 = 3; else if (Get.data[1] > (0.85*readings2)) on_num2 = 4; else if (Get.data[1] > (0.8*readings2)) on_num2 = 5; else if (Get.data[1] > (0.7*readings2)) on_num2 = 6; else if (Get.data[1] > (0.6*readings2)) on_num2 = 7; else on_num2 = 8; if((dy2 > -1)&& (dty2 < 0)) on_num2 += 2; } else { if (dy2 < 0 ) on_num2 = 0; else if (Get.data[1] > (0.99*readings2)) on_num2 = 0; else if (Get.data[1] > (0.96*readings2)) on_num2 = 0; else if (Get.data[1] > (0.9*readings2)) on_num2 = 1; else if (Get.data[1] > (0.85*readings2)) on_num2 = 2; else if (Get.data[1] > (0.8*readings2)) on_num2 = 3; else if (Get.data[1] > (0.7*readings2)) on_num2 = 4; else if (Get.data[1] > (0.6*readings2)) on_num2 = 5; else on_num2 = 7; if((dy2 > -1)&& (dty2 < 0))

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on_num2 += 2; } //on_num2 = 16; go_num2++; go_num2 = go_num2 % 16; if ((go_num2 < on_num2) &&(onandoff2)) // if (onandoff2) { word2=0x02; SetCtrlVal (panelHandle, PANEL_LED2, ON); } else { word2=0x00; SetCtrlVal (panelHandle, PANEL_LED2, OFF); } // number 3 if(readings3 > 500) { if (dy3 < 0 ) on_num3 = 0; else if (Get.data[2] > (0.999*readings3)) on_num3 = 1; else if (Get.data[2] > (0.998*readings3)) on_num3 = 3; /* else if (Get.data[2] > (0.997*readings3)) on_num3 = 5; // else if (Get.data[2] > (0.996*readings3)) on_num3 = 7; // else if (Get.data[2] > (0.994*readings3)) on_num3 = 9; // else if (Get.data[2] > (0.992*readings3)) on_num3 = 10; else if (Get.data[2] > (0.99*readings3)) on_num3 = 13; */ else if (Get.data[2] > (0.96*readings3)) on_num3 = 10; /* else if (Get.data[2] > (0.9*readings3)) on_num3 = 13; else if (Get.data[2] > (0.75*readings3)) on_num3 = 14; */ else on_num3 = 16; if((dy3 < 10.0) && (dy3 > -5)) { if (dty3 < -1.0) on_num3 += 16; else if (dty3 < -0.7) on_num3 += 12; else if (dty3 < -0.2) on_num3 += 8; } } else if(readings3 > 300) { if (dy3 < 0 ) on_num3 = 0; else if (Get.data[2] > (0.995*readings3)) on_num3 = 2; else if (Get.data[2] > (0.99*readings3)) on_num3 = 3; else if (Get.data[2] > (0.96*readings3)) on_num3 = 3;

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else if (Get.data[2] > (0.9*readings3)) on_num3 = 4; else if (Get.data[2] > (0.85*readings3)) on_num3 = 4; else if (Get.data[2] > (0.8*readings3)) on_num3 = 5; else if (Get.data[2] > (0.7*readings3)) on_num3 = 6; else if (Get.data[2] > (0.6*readings3)) on_num3 = 7; else on_num3 = 8; if((dy3 < 80.0) && (dy3 > -10)) { if (dty3 < -2.0) on_num3 += 6; else if (dty3 < -0.7) on_num3 += 4; else if (dty3 < -0.2) on_num3 += 3; else if (dty3 < 0) on_num3 += 2; } } else if ( readings3 > 150) { if (dy3 < 0 ) on_num3 = 0; else if (Get.data[2] > (0.99*readings3)) on_num3 = 0; else if (Get.data[2] > (0.96*readings3)) on_num3 = 2; else if (Get.data[2] > (0.9*readings3)) on_num3 = 3; else if (Get.data[2] > (0.85*readings3)) on_num3 = 4; else if (Get.data[2] > (0.8*readings3)) on_num3 = 5; else if (Get.data[2] > (0.7*readings3)) on_num3 = 6; else if (Get.data[2] > (0.6*readings3)) on_num3 = 7; else on_num3 = 8; if((dy3 > -1)&& (dty3 < 0)) on_num3 += 2; } else { if (dy3 < 0 ) on_num3 = 0; else if (Get.data[2] > (0.99*readings3)) on_num3 = 0; else if (Get.data[2] > (0.96*readings3)) on_num3 = 0; else if (Get.data[2] > (0.9*readings3)) on_num3 = 1; else if (Get.data[2] > (0.85*readings3)) on_num3 = 2; else if (Get.data[2] > (0.8*readings3)) on_num3 = 3; else if (Get.data[2] > (0.7*readings3)) on_num3 = 4; else if (Get.data[2] > (0.6*readings3)) on_num3 = 5; else on_num3 = 7; if((dy3 > -1)&& (dty3 < 0)) on_num3 += 2; } //on_num3 = 16;

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go_num3++; go_num3 = go_num3 % 16; if ((go_num3 < on_num3) &&(onandoff3)) // if (onandoff3) { word3=0x04; SetCtrlVal (panelHandle, PANEL_LED3, ON); } else { word3=0x00; SetCtrlVal (panelHandle, PANEL_LED3, OFF); } // number 4 if(readings4 > 500) { if (dy4 < 0 ) on_num4 = 0; else if (Get.data[3] > (0.999*readings4)) on_num4 = 1; else if (Get.data[3] > (0.998*readings4)) on_num4 = 3; /* else if (Get.data[3] > (0.997*readings4)) on_num4 = 6; // else if (Get.data[3] > (0.996*readings4)) on_num4 = 8; // else if (Get.data[3] > (0.994*readings4)) on_num4 = 9; // else if (Get.data[3] > (0.992*readings4)) on_num4 = 10; else if (Get.data[3] > (0.99*readings4)) on_num4 = 11; */ else if (Get.data[3] > (0.95*readings4)) on_num4 = 10; /* else if (Get.data[3] > (0.9*readings4)) on_num4 = 13; else if (Get.data[3] > (0.75*readings4)) on_num4 = 14; */ else on_num4 = 16; if((dy4 < 10.0) && (dy4 > -5)) { if (dty4 < -1.0) on_num4 += 16; else if (dty4 < -0.7) on_num4 += 12; else if (dty4 < -0.2) on_num4 += 8; } } else if(readings4 > 300) { if (dy4 < 0 ) on_num4 = 0; else if (Get.data[3] > (0.995*readings4)) on_num4 = 2; else if (Get.data[3] > (0.99*readings4)) on_num4 = 3; else if (Get.data[3] > (0.96*readings4)) on_num4 = 3; else if (Get.data[3] > (0.9*readings4)) on_num4 = 4; else if (Get.data[3] > (0.85*readings4)) on_num4 = 4; else if (Get.data[3] > (0.8*readings4)) on_num4 = 5;

213

else if (Get.data[3] > (0.7*readings4)) on_num4 = 6; else if (Get.data[3] > (0.6*readings4)) on_num4 = 7; else on_num4 = 8; if((dy4 < 80.0) && (dy4 > -10)) { if (dty4 < -2.0) on_num4 += 6; else if (dty4 < -0.7) on_num4 += 4; else if (dty4 < -0.2) on_num4 += 3; else if (dty4 < 0) on_num4 += 2; } } else if ( readings4 > 150) { if (dy4 < 0 ) on_num4 = 0; else if (Get.data[3] > (0.99*readings4)) on_num4 = 0; else if (Get.data[3] > (0.96*readings4)) on_num4 = 2; else if (Get.data[3] > (0.9*readings4)) on_num4 = 3; else if (Get.data[3] > (0.85*readings4)) on_num4 = 4; else if (Get.data[3] > (0.8*readings4)) on_num4 = 5; else if (Get.data[3] > (0.7*readings4)) on_num4 = 6; else if (Get.data[3] > (0.6*readings4)) on_num4 = 7; else on_num4 = 8; if((dy4 > -1)&& (dty4 < 0)) on_num4 += 2; } else { if (dy4 < 0 ) on_num4 = 0; else if (Get.data[3] > (0.99*readings4)) on_num4 = 0; else if (Get.data[3] > (0.96*readings4)) on_num4 = 0; else if (Get.data[3] > (0.9*readings4)) on_num4 = 1; else if (Get.data[3] > (0.85*readings4)) on_num4 = 2; else if (Get.data[3] > (0.8*readings4)) on_num4 = 3; else if (Get.data[3] > (0.7*readings4)) on_num4 = 4; else if (Get.data[3] > (0.6*readings4)) on_num4 = 5; else on_num4 = 7; if((dy4 > -1)&& (dty4 < 0)) on_num4 += 2; } //on_num4 = 16; go_num4++; go_num4 = go_num4 % 16; if ((go_num4 < on_num4) &&(onandoff4))

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// if (onandoff4) { word4=0x08; SetCtrlVal (panelHandle, PANEL_LED4, ON); } else { word4=0x00; SetCtrlVal (panelHandle, PANEL_LED4, OFF); } // number 5 if(readings5 > 500) { if (dy5 < 0 ) on_num5 = 0; else if (Get.data[4] > (0.999*readings5)) on_num5 = 1; else if (Get.data[4] > (0.998*readings5)) on_num5 = 3; /* else if (Get.data[4] > (0.997*readings5)) on_num5 = 5; // else if (Get.data[4] > (0.996*readings5)) on_num5 = 7; // else if (Get.data[4] > (0.994*readings5)) on_num5 = 9; // else if (Get.data[4] > (0.992*readings5)) on_num5 = 10; else if (Get.data[4] > (0.99*readings5)) on_num5 = 11; */ else if (Get.data[4] > (0.95*readings5)) on_num5 = 10; /* else if (Get.data[4] > (0.9*readings5)) on_num5 = 13; else if (Get.data[4] > (0.75*readings5)) on_num5 = 14; */ else on_num5 = 16; if((dy5 < 10.0) && (dy5 > -5)) { if (dty5 < -1.0) on_num5 += 16; else if (dty5 < -0.7) on_num5 += 12; else if (dty5 < -0.2) on_num5 += 8; } } else if(readings5 > 300) { if (dy5 < 0 ) on_num5 = 0; else if (Get.data[4] > (0.995*readings5)) on_num5 = 2; else if (Get.data[4] > (0.99*readings5)) on_num5 = 3; else if (Get.data[4] > (0.96*readings5)) on_num5 = 3; else if (Get.data[4] > (0.9*readings5)) on_num5 = 4; else if (Get.data[4] > (0.85*readings5)) on_num5 = 4; else if (Get.data[4] > (0.8*readings5)) on_num5 = 5; else if (Get.data[4] > (0.7*readings5)) on_num5 = 6; else if (Get.data[4] > (0.6*readings5)) on_num5 = 7; else on_num5 = 8;

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if((dy5 < 80.0) && (dy5 > -10)) { if (dty5 < -2.0) on_num5 += 6; else if (dty5 < -0.7) on_num5 += 4; else if (dty5 < -0.2) on_num5 += 3; else if (dty5 < 0) on_num5 += 2; } } else if (readings5 > 150) { if (dy5 < 0 ) on_num5 = 0; else if (Get.data[4] > (0.99*readings5)) on_num5 = 0; else if (Get.data[4] > (0.96*readings5)) on_num5 = 2; else if (Get.data[4] > (0.9*readings5)) on_num5 = 3; else if (Get.data[4] > (0.85*readings5)) on_num5 = 4; else if (Get.data[4] > (0.8*readings5)) on_num5 = 5; else if (Get.data[4] > (0.7*readings5)) on_num5 = 6; else if (Get.data[4] > (0.6*readings5)) on_num5 = 7; else on_num5 = 8; if((dy5 > -1)&& (dty5 < 0)) on_num5 += 2; } else { if (dy5 < 0 ) on_num5 = 0; else if (Get.data[4] > (0.99*readings5)) on_num5 = 0; else if (Get.data[4] > (0.96*readings5)) on_num5 = 0; else if (Get.data[4] > (0.9*readings5)) on_num5 = 1; else if (Get.data[4] > (0.85*readings5)) on_num5 = 2; else if (Get.data[4] > (0.8*readings5)) on_num5 = 3; else if (Get.data[4] > (0.7*readings5)) on_num5 = 4; else if (Get.data[4] > (0.6*readings5)) on_num5 = 5; else on_num5 = 7; if((dy5 > -1)&& (dty5 < 0)) on_num5 += 2; } //on_num5 = 16; go_num5++; go_num5 = go_num5 % 16; if ((go_num5 < on_num5) &&(onandoff5)) // if (onandoff5)

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{ word5=0x10; SetCtrlVal (panelHandle, PANEL_LED5, ON); } else { word5=0x00; SetCtrlVal (panelHandle, PANEL_LED5, OFF); } // number 6 if(readings6 > 500) { if (dy6 < 0 ) on_num6 = 0; else if (Get.data[5] > (0.999*readings6)) on_num6 = 1; else if (Get.data[5] > (0.998*readings6)) on_num6 = 3; /* else if (Get.data[5] > (0.997*readings6)) on_num6 = 5; else if (Get.data[5] > (0.996*readings6)) on_num6 = 7; else if (Get.data[5] > (0.994*readings6)) on_num6 = 9; else if (Get.data[5] > (0.992*readings6)) on_num6 = 10; else if (Get.data[5] > (0.99*readings6)) on_num6 = 11; */ else if (Get.data[5] > (0.95*readings6)) on_num6 = 10; /* else if (Get.data[5] > (0.9*readings6)) on_num6 = 13; else if (Get.data[5] > (0.75*readings6)) on_num6 = 14; */ else on_num6 = 16; if((dy6 < 10.0) && (dy6 > -5)) { if (dty6 < -1.0) on_num6 += 16; else if (dty6 < -0.7) on_num6 += 12; else if (dty6 < -0.2) on_num6 += 8; } } else if(readings6 > 300) { if (dy6 < 0 ) on_num6 = 0; else if (Get.data[5] > (0.995*readings6)) on_num6 = 2; else if (Get.data[5] > (0.99*readings6)) on_num6 = 3; else if (Get.data[5] > (0.96*readings6)) on_num6 = 3; else if (Get.data[5] > (0.9*readings6)) on_num6 = 4; else if (Get.data[5] > (0.85*readings6)) on_num6 = 4; else if (Get.data[5] > (0.8*readings6)) on_num6 = 5; else if (Get.data[5] > (0.7*readings6)) on_num6 = 6; else if (Get.data[5] > (0.6*readings6)) on_num6 = 7; else on_num6 = 8;

217

if((dy6 < 80.0) && (dy6 > -10)) { if (dty6 < -2.0) on_num6 += 6; else if (dty6 < -0.7) on_num6 += 4; else if (dty6 < -0.2) on_num6 += 3; else if (dty6< 0) on_num6 += 2; } } else if ( readings6 > 150) { if (dy6 < 0 ) on_num6 = 0; else if (Get.data[5] > (0.99*readings6)) on_num6 = 0; else if (Get.data[5] > (0.96*readings6)) on_num6 = 2; else if (Get.data[5] > (0.9*readings6)) on_num6 = 3; else if (Get.data[5] > (0.85*readings6)) on_num6 = 4; else if (Get.data[5] > (0.8*readings6)) on_num6 = 5; else if (Get.data[5] > (0.7*readings6)) on_num6 = 6; else if (Get.data[5] > (0.6*readings6)) on_num6 = 7; else on_num6 = 8; if((dy6 > -1)&& (dty6 < 0)) on_num6 += 2; } else { if (dy6 < 0 ) on_num6 = 0; else if (Get.data[5] > (0.99*readings6)) on_num6 = 0; else if (Get.data[5] > (0.96*readings6)) on_num6 = 0; else if (Get.data[5] > (0.9*readings6)) on_num6 = 1; else if (Get.data[5] > (0.85*readings6)) on_num6 = 2; else if (Get.data[5] > (0.8*readings6)) on_num6 = 3; else if (Get.data[5] > (0.7*readings6)) on_num6 = 4; else if (Get.data[5] > (0.6*readings6)) on_num6 = 5; else on_num6 = 7; if((dy6 > -1)&& (dty6 < 0)) on_num6 += 2; } // on_num6 = 16; go_num6++; go_num6 = go_num6 % 16; if ((go_num6 < on_num6) &&(onandoff6)) // if (onandoff6) {

218

word6=0x20; SetCtrlVal (panelHandle, PANEL_LED6, ON); } else { word6=0x00; SetCtrlVal (panelHandle, PANEL_LED6, OFF); } GetCtrlVal (panelHandle, PANEL_FAN, &fan); if (fan) word7=0x40; else word7= 0x00; /* word5=word6>>1; word4=word6>>2; word3=word6>>3; word2=word6>>4; word1=word6>>5; */ //sent_word = 0x42; if (Get.data[9]>80) { sent_word = 0x40; SetCtrlVal (panelHandle, PANEL_ALARM, ON); SetCtrlVal (panelHandle, PANEL_LED1, OFF); SetCtrlVal (panelHandle, PANEL_LED2, OFF); SetCtrlVal (panelHandle, PANEL_LED3, OFF); SetCtrlVal (panelHandle, PANEL_LED4, OFF); SetCtrlVal (panelHandle, PANEL_LED5, OFF); SetCtrlVal (panelHandle, PANEL_LED6, OFF); } else { sent_word = word1 | word2 | word3 | word4|word5|word6|word7; SetCtrlVal (panelHandle, PANEL_ALARM, OFF); } Fmt(sent_word1,"%s<%i %i %i %i %i %i %i %i",sent_word, on_num1,on_num2,on_num3,on_num4,on_num5,on_num6,go_num6); SetCtrlVal (panelHandle, PANEL_TEST, sent_word1); outp(LPT1,sent_word); } int CVICALLBACK Change_display (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: break; } return 0; } int CVICALLBACK set_wave (int panel, int control, int event,

219

void *callbackData, int eventData1, int eventData2) { double wave; char TEMPSTR[7]; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_RING1, &wave); SetCtrlVal(setHandle, SET_SETPOINT, decalib(wave)); break; } return 0; } int CVICALLBACK set_wave1 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { double wave; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_RING2, &wave); SetCtrlVal(setHandle, SET_SETPOINT_2, decalib(wave)); break; } return 0; } int CVICALLBACK set_wave2 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { double wave; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_RING3, &wave); SetCtrlVal(setHandle, SET_SETPOINT_3, decalib(wave)); break; } return 0; } int CVICALLBACK set_wave3 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { double wave; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_RING4, &wave); SetCtrlVal(setHandle, SET_SETPOINT_4, decalib(wave)); break; }

220

return 0; } int CVICALLBACK set_wave4 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { double wave; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_RING5, &wave); SetCtrlVal(setHandle, SET_SETPOINT_5, decalib(wave)); break; } return 0; } int CVICALLBACK set_wave5 (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { double wave; switch (event) { case EVENT_COMMIT: GetCtrlVal (setHandle, SET_RING6, &wave); SetCtrlVal(setHandle, SET_SETPOINT_6, decalib(wave)); break; } return 0; } int CVICALLBACK timer3_control (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { char save_fmt[30]; char sent_word2[200]; switch (event) { case EVENT_TIMER_TICK: // SetSystemAttribute (ATTR_ALLOW_UNSAFE_TIMER_EVENTS, 1); if (goflag2 ==1) { // Fmt(sent_word2,"%s<%f[p50]",Rsbuf); SetCtrlVal (panelHandle, PANEL_TEST_2, Rsbuf); if (choice1) { Fmt(buf, "%s<%i: %f[p2] %f[p2] %f[p2] %f[p2] : %f[p2] %f[p2] %f[p2]", datanum, Get.data[0], Get.data[1],Get.data[2],Get.data[3],Get.data[4],Get.data[5],Get.data[6]);

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} else {

Fmt(buf, "%s<%i: %f[p2] %f[p2] %f[p2] %f[p2] %f[p2] %f[p2] : %f[p2] %f[p2] %f[p2] %f[p2] %f[p2] %f[p2] %f[p2] ", datanum, Get.data[0], Get.data[1],Get.data[2],Get.data[3],Get.data[4],Get.data[5],Get.data[6], Get.data[7],Get.data[8],Get.data[9],Get.data[10],Get.data[11],Get.data[12]);

} InsertListItem (panelHandle, PANEL_TEMP_LIST, -1, buf, 0); GetNumListItems (panelHandle, PANEL_TEMP_LIST , &index_num); SetCtrlIndex (panelHandle, PANEL_TEMP_LIST, index_num-1); GetCtrlVal (graphHandle, GRAPH_BINARYSWITCH, &read); graphdata7[0] = Get.data[0]; graphdata7[1] = Get.data[2]; graphdata7[2] = Get.data[3]; graphdata7[3] = Get.data[9]; graphdata7[4] = Get.data[8]; if (read == 0) { graphdata1[0] = calib(Get.data[0]); graphdata2[0] = calib(Get.data[1]); graphdata3[0] = calib(Get.data[2]); graphdata4[0] = calib(Get.data[3]); graphdata5[0] = calib(Get.data[4]); graphdata6[0] = calib(Get.data[5]); graphdata1[1] = calib(readings1); graphdata2[1] = calib(readings2); graphdata3[1] = calib(readings3); graphdata4[1] = calib(readings4); graphdata5[1] = calib(readings5); graphdata6[1] = calib(readings6); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1_1,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2_1,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3_1,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4_1,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5_1,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6_1,ATTR_VISIBLE,1); if (choice1) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6_1,ATTR_VISIBLE,0);

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} if(dim1) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH1_1, graphdata1, 2, 0, 0,VAL_DOUBLE); if(dim2) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH2_1, graphdata2, 2, 0, 0,VAL_DOUBLE); if(dim3) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH3_1, graphdata3, 2, 0, 0,VAL_DOUBLE); if(dim4) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH4_1, graphdata4, 2, 0, 0,VAL_DOUBLE); if(dim5) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH5_1, graphdata5, 2, 0, 0,VAL_DOUBLE); if(dim6) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH6_1, graphdata6, 2, 0, 0,VAL_DOUBLE); PlotStripChart (panelHandle, PANEL_TEMP_GRAPH7, graphdata7, 2, 0, 0,VAL_DOUBLE); /*Kadir added if(dim7) - */ } else { graphdata1[0] = Get.data[0]; graphdata2[0] = Get.data[1]; graphdata3[0] = Get.data[2]; graphdata4[0] = Get.data[3]; graphdata5[0] = Get.data[4]; graphdata6[0] = Get.data[5]; graphdata1[1] = readings1; graphdata2[1] = readings2; graphdata3[1] = readings3; graphdata4[1] = readings4; graphdata5[1] = readings5; graphdata6[1] = readings6; SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5,ATTR_VISIBLE,1); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6,ATTR_VISIBLE,1); if (choice1) { SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5,ATTR_VISIBLE,0);

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SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6,ATTR_VISIBLE,0); } SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH1_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH2_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH3_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH4_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH5_1,ATTR_VISIBLE,0); SetCtrlAttribute(graphHandle,GRAPH_TEMP_GRAPH6_1,ATTR_VISIBLE,0); if(dim1) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH1, graphdata1, 2, 0, 0,VAL_DOUBLE); if(dim2) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH2, graphdata2, 2, 0, 0,VAL_DOUBLE); if(dim3) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH3, graphdata3, 2, 0, 0,VAL_DOUBLE); if(dim4) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH4, graphdata4, 2, 0, 0,VAL_DOUBLE); if(dim5) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH5, graphdata5, 2, 0, 0,VAL_DOUBLE); if(dim6) PlotStripChart (graphHandle, GRAPH_TEMP_GRAPH6, graphdata6, 2, 0, 0,VAL_DOUBLE); PlotStripChart (panelHandle, PANEL_TEMP_GRAPH7, graphdata7, 5, 0, 0,VAL_DOUBLE); } datapoints[0][datanum] = Get.data[0]; datapoints[1][datanum] = Get.data[1]; datapoints[2][datanum] = Get.data[2]; datapoints[3][datanum] = Get.data[3]; datapoints[4][datanum] = Get.data[4]; datapoints[5][datanum] = Get.data[5]; datapoints[6][datanum] = Get.data[6]; datapoints[7][datanum] = Get.data[7]; datapoints[8][datanum] = Get.data[8]; datapoints[9][datanum] = Get.data[9]; datapoints[10][datanum] = Get.data[10]; datapoints[11][datanum] = Get.data[11]; datapoints[12][datanum] = Get.data[12]; GetCtrlVal (panelHandle, PANEL_HEATING, &sign); GetCtrlVal (panelHandle, PANEL_COMPARISON, &save1); if (sign == 1) { sum=Get.data[4]-datapoints[4][0]; save=save + sum; Fmt(save_fmt,"%s<%f[p2]",save); SetCtrlVal (panelHandle, PANEL_TEST2, save_fmt); } if ((save1 !=0)&&((save1 - save)<1))

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{ goflag=0; goflag1=0; goflag2=0; SetCtrlVal (panelHandle, PANEL_HEATING,0); //SetCtrlAttribute(panelHandle,PANEL_STOPBUTTON,ATTR_VISIBLE,1); //SetCtrlAttribute(panelHandle,PANEL_GOBUTTON,ATTR_VISIBLE,0); } datanum++; SetCtrlVal (panelHandle, PANEL_LED, OFF); } break; } return 0; } int CVICALLBACK fan_control (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: if (end) { GetCtrlVal (panelHandle, PANEL_FAN, &fan); if (fan) word=0x40; else word= 0x00; outp(LPT1,word); } else break; } return 0; } int CVICALLBACK close_graph (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: HidePanel (graphHandle); break; } return 0; } int CVICALLBACK display_graph (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: DisplayPanel (graphHandle); break; } return 0; }

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Vita Kathiravan Krishnamurthy

EDUCATION

• Pennsylvania State University, University Park, PA (2002-2005). o Doctor of Philosophy in Agricultural & Biological Engineering (Expected:

Summer 2006) - GPA 3.73/4.00. o Master of Science in Agricultural & Biological Engineering - GPA

3.55/4.00. • Tamil Nadu Agricultural University, Tamil Nadu, India (1995-1999).

o Bachelor of Engineering in Agricultural Engineering - GPA 8.84/10.00. SELECTED AWARDS AND HONORS

• Annual graduate exhibition, Pennsylvania State University • Third place, 2005; First place, 2003

• Annual College of Agricultural Sciences, Gamma Sigma Delta Undergraduate and Graduate Research Expo, Pennsylvania State University

• Third place, 2005; First place, 2004; Gerald T. Gentry award, 2002 • “The Chancellor’s list®”, Educational Communications Inc., 2005. • Graduate school teaching certificate in recognition of a series of college teaching

experiences, Pennsylvania State University, summer 2004. • Outstanding paper presentation award, Evans family lecture for graduate research,

College of Agricultural Sciences, Pennsylvania State University, April 14, 2004. PEER REVIEWED PUBLICATIONS

• Krishnamurthy, K., A. Demirci, V.M. Puri, and C.N. Cutter. 2004. Effect of packaging materials on inactivation of pathogenic microorganisms on meat during irradiation, Transactions of ASAE. 47: 1141-1149.

• Krishnamurthy, K., A. Demirci, and J. Irudayaraj. 2004. Inactivation of Staphylococcus aureus by pulsed UV-light treatment. Journal of Food Protection. 67: 1027-1030.

• Krishnamurthy, K. and A. Demirci. 2006. Disinfection of water through flow-through ultraviolet light disinfection system. Ultrapure Water (In review)

• Krishnamurthy, K., A. Demirci, and J. Irudayaraj. 2006. Staphylococcus aureus inactivation in milk and milk foam by pulsed UV-light treatment. Journal of Food Science (In preparation).

• Krishnamurthy, K., A. Demirci, and J. Irudayaraj. 2006. Continuous milk treatment using pulsed UV-light for inactivation of Staphylococcus aureus. International Journal of Food Microbiology (In preparation).

• Krishnamurthy, K., S. Jun, J. Irudayaraj, and A. Demirci. 2006. Infrared heat treatment for inactivation of Staphylococcus aureus in milk. International Journal of Food microbiology (In preparation).

• Krishnamurthy, K., J.C. Tewari, A. Demirci, and J. Irudayaraj.2006. Spectroscopic and microscopic investigations of inactivation of Staphyloccoccus aureus by pulsed UV-light and infrared heating. Applied and Environmental Microbiology (In preparation).

Decontamination of Milk and Water by Pulsed UV-light and Infrared Heating

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